HK1170250B

HK1170250B - Chimeric influenza virus-like particles comprising hemagglutinin

Info

Publication number: HK1170250B
Application number: HK12111105.5A
Authority: HK
Inventors: 马农．科图雷; 米凯莱．达吉斯; 皮埃尔-奥列弗．拉瓦; 路易斯-菲利普．韦齐纳; 马克-安德烈．德奥斯特
Original assignee: 麦迪卡格公司
Priority date: 2009-06-24
Filing date: 2010-06-25
Publication date: 2015-10-02

Description

Chimeric influenza virus-like particles comprising hemagglutinin

Technical Field

This application claims priority to U.S. provisional application No.61/220,161 filed on 24.6.2009.

The present invention relates to virus-like particles. More particularly, the present invention relates to virus-like particles comprising chimeric influenza hemagglutinin and methods of producing chimeric influenza virus-like particles.

Background

Influenza is the leading cause of human death due to respiratory viruses, and in the influenza season, it is estimated that 10-20% of the world population is infected, resulting in 250-500,000 deaths each year.

The current approach to combat human influenza is annual vaccination. Vaccines are usually a combination of several strains that are predicted to be virulent strains (dominant strains) in the upcoming influenza season, but the number of vaccine agents produced annually is insufficient to vaccinate the world population. For example, canada and the united states, based on current yields, obtain enough vaccine agents to immunize about one-third of their population, while only 17% of european populations are eligible for vaccines — this yield is not sufficient when a worldwide influenza pandemic comes. Even though the required annual yield in a given year may be achieved in some way, the virulent strains change from year to year, and so large reserves of low demand time in a year are not practical. The economic, large-scale production of effective influenza vaccines is of great concern to governments and private enterprises alike.

The influenza Hemagglutinin (HA) surface glycoprotein is a receptor binding protein and is also a membrane fusion protein. It is a trimer of identical subunits, each subunit comprising two disulfide-linked polypeptides HA1 and HA2, which result from proteolytic cleavage of the precursor HA0, with a signal peptide sequence at the N-terminus of HA0 and a membrane anchoring sequence at the C-terminus of HA 0. Cleavage to form HA1 and HA2 yielded the N-terminus of the smaller polypeptide HA2, with the C-terminus of HA2 having a membrane anchoring sequence. Cleavage is necessary for membrane fusion activity, but not for immunogenicity. The N-terminal sequence of HA2 is called a "fusion peptide" because cleavage of a similar hydrophobic sequence is also required for other viral fusion proteins, and a synthetic peptide analog of 20 residues of this sequence is fused to the membrane in vitro.

In general, the surface of the spherical "head" comprises several flexible loops with well-characterized and variable antigenic regions, designated sites A, B, C, D and E (reviewed in Wiley et al, 1987, Annu Rev Biochem 56: 365-. The insertion or substitution of short peptide sequences at some sites (e.g., B and E) for immunological studies has been described (Garcia-Sastre et al 1995 Biologicals 23: 171-. The Fc domain of Epidermal Growth Factor (EGF), single chain antibody (scFV) and IgG, ranging in size from 53 to 246 amino acids, has been inserted at the N-terminus of H7 and chimeras have been successfully expressed (Hatziioannou et al, 1999 Human Gene Therapy 10: 1533-1544). Recently, domains of 90 and 140 amino acids of the protective antigen of Bacillus anthracis (Bacillus anthracalis) have been fused to the amino terminus of H3 (Li et al, 2005.J.Virol 79: 10003-Asahando 1002). Copeland (Copeland et al, 2005.J. Virol 79: 6459-6471) describes the expression of gp120Env HIV surface glycoprotein on the H3 stalk (talk) where the gp120 domain replaces the entire globular head of HA.

Several recombinant products have been developed as candidate recombinant influenza vaccines. These approaches focus on the expression, production and purification of influenza A HA and NA proteins, including the expression of these proteins by baculovirus-infected insect cells (Crawford et al, 1999 Vaccine 17: 2265-74; Johansson, 1999 Vaccine 17: 2073-80), viral vectors and DNA Vaccine constructs (Olsen et al, 1997 Vaccine 15: 1149-56).

The production of non-infectious influenza virus strains for vaccine purposes is one way to avoid accidental infections. As an alternative, virus-like particles (VLPs) have been investigated as an alternative to cultured viruses. VLPs mimic the structure of the viral capsid, but lack the genome and therefore cannot replicate or provide a tool for reinfection. Current techniques for the production of influenza VLPs rely on the co-expression of multiple viral proteins, and this dependence is a disadvantage of these techniques, since in the case of pandemics and annual epidemics, response time is crucial for vaccination. A simpler VLP production system (e.g. relying on the expression of only one or a few viral proteins without the need to express non-structural viral proteins) is needed to accelerate vaccine development.

Enveloped viruses can obtain a lipid envelope when "budding" from an infected cell, and membranes from the plasma membrane or membranes of internal organelles. For example, in mammalian cells or baculovirus cell systems, influenza viruses bud from the plasma membrane (Quan et al 2007J. Virol 81: 3514-3524). Only a few enveloped viruses are known to infect plants (e.g., members of the tomato spotted wilt virus (Tospovirus) and Rhabdovirus (Rhabdovirus)). Known plant enveloped viruses are characterized by budding from the inner membrane of the host cell, rather than from the plasma membrane. Although a small number of recombinant VLPs have been produced in plant hosts, none of them are derived from the plasma membrane, which raises the question of whether plasma membrane-derived VLPs, including influenza VLPs, can be produced in plants.

The formation of VLPs in any system is highly demanding on protein structure-altering the short sequences of the surface loops corresponding to the selected globular structure may not have much impact on the expression of the polypeptide itself, but structural studies demonstrating that these alterations affect VLP formation are lacking. The coordination and structure of multiple regions of HA (e.g., membrane anchor sequences, trimeric stem or stalk regions separating the globular head from the membrane) evolves with the virus and may not be able to make similar changes without loss of HA trimeric integrity and VLP formation.

In WO 2009/009876, the inventors have described the production of influenza HAVLP.

Disclosure of Invention

The present invention relates to virus-like particles. More particularly, the present invention relates to virus-like particles comprising chimeric influenza hemagglutinin and methods of producing chimeric influenza hemagglutinin virus-like particles.

It is an object of the present invention to provide improved chimeric influenza Virus Like Particles (VLPs).

The present invention provides a polypeptide comprising a chimeric influenza HA comprising a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC) and a Transmembrane Domain Cluster (TDC), wherein: SDC comprises F '1, F' 2, and F subdomains; HDC comprises RB, E1, and E2 subdomains; TDC comprises TmD and Ctail subdomain; and wherein at least one subdomain is from a first influenza HA and the other subdomains are from one or more second influenza HA. The first and second influenza HA may be independently selected from H1, H3, H5, and B. Furthermore, the polypeptide may comprise a signal peptide.

The invention also provides a nucleic acid encoding a polypeptide comprising a chimeric influenza HA comprising a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC), and a Transmembrane Domain Cluster (TDC), wherein: SDC comprises F '1, F' 2, and F subdomains; HDC comprises RB, E1, and E2 subdomains; TDC comprises TmD and Ctail subdomain; and wherein at least one subdomain is from a first influenza HA and the other subdomains are from one or more second influenza HA. The nucleic acid may also encode a polypeptide comprising a signal peptide in addition to the SDC, HDC and TDC.

Also provided is a method of producing chimeric influenza Virus Like Particles (VLPs) in a plant, the method comprising:

a) introducing into a plant or portion thereof a nucleic acid encoding a chimeric influenza HA comprising a signal peptide, a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC), and a Transmembrane Domain Cluster (TDC), wherein: SDC comprises F '1, F' 2, and F subdomains; HDC comprises RB, E1, and E2 subdomains; TDC comprises TmD and Ctail subdomain; and wherein at least one subdomain is from a first influenza HA and the other subdomains are from one or more second influenza HA, and

b) incubating the plant or portion thereof under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present invention includes the above method, wherein in the step of introducing (step a), the nucleic acid is introduced into the plant in a transient manner. Alternatively, in the step of introducing (step a), the nucleic acid is introduced into the plant and stably integrated. The method may further comprise the steps of: c) the host was harvested and the VLPs were purified.

The present invention provides a plant or part thereof comprising a chimeric influenza HA comprising a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC) and a Transmembrane Domain Cluster (TDC), or a nucleic acid sequence encoding a chimeric influenza HA, wherein: SDC comprises F '1, F' 2, and F subdomains; HDC comprises RB, E1, and E2 subdomains; TDC comprises TmD and Ctail subdomain; and wherein at least one subdomain is from a first influenza HA and the other domains are from one or more second influenza HA.

The plant or part thereof may also comprise a nucleic acid comprising a nucleotide sequence encoding one or more than one chaperone protein operably linked to a regulatory region active in plants. The one or more chaperones may be selected from Hsp40 and Hsp 70.

The present invention relates to a virus-like particle (VLP) comprising a chimeric influenza HA comprising a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC) and a Transmembrane Domain Cluster (TDC), wherein: SDC comprises F '1, F' 2, and F subdomains; HDC comprises RB, E1, and E2 subdomains; TDC comprises TmD and Ctail subdomain; and wherein at least one subdomain is from a first influenza HA and the other domains are from one or more second influenza HA. The VLP may further comprise plant-specific N-glycans or modified N-glycans.

Also provided are compositions comprising an effective dose of the aforementioned VLPs and a pharmaceutically acceptable carrier.

In another aspect of the invention, there is provided a method of inducing immunity to an influenza virus infection in a subject, comprising administering to the subject a VLP. VLPs may be administered to a subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

Regulatory regions operably linked to sequences encoding the chimeric HA proteins include regulatory regions effective in plant cells, insect cells, or yeast cells. Such regulatory regions may include a plastocyanin regulatory region, a ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) regulatory region, a chlorophyll a/b binding protein (CAB) regulatory region, or an ST-LS1 regulatory region. Other regulatory regions include 5 'UTR, 3' UTR or terminator sequences. The plastocyanin regulatory region can be alfalfa plastocyanin regulatory region; the 5 'UTR, 3' UTR or terminator sequence may also be an alfalfa sequence.

The invention provides a chimeric influenza HA polypeptide consisting of a first influenza and a second influenza, the first and second influenza can be independently selected from B, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16; provided that the first and second influenza are not of the same influenza type, subtype or have not the same origin.

According to some aspects of the invention, the chimeric influenza HA polypeptide comprises a signal peptide sequence that may be selected from the group consisting of a native signal peptide sequence, an alfalfa PDI signal peptide sequence, an influenza H5 signal peptide sequence, and an influenza H1 signal peptide sequence.

The present invention provides a method of producing a VLP comprising a chimeric influenza Hemagglutinin (HA) in a host (including a plant, insect or yeast) capable of producing the VLP, comprising introducing into the host a nucleic acid encoding the chimeric influenza HA comprising a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC) and a Transmembrane Domain Cluster (TDC), and incubating the host under conditions which allow expression of the nucleic acid, thereby producing the VLP, wherein: SDC comprises F '1, F' 2, and F subdomains; HDC comprises RB, E1, and E2 subdomains; TDC comprises TmD and Ctail subdomain; and wherein at least one subdomain is from a first influenza HA and the other domains are from one or more second influenza HA.

The production of these particles in plants has several advantages over the production of VLPs in insect cell cultures. Plant lipids can stimulate specific immune cells and enhance the induced immune response. The membranes of plants are composed of lipids, Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE), and also contain glycosphingolipids characteristic of plants and some bacteria and protozoa. Sphingolipids are not common because they are not glycerides (e.g. PC or PE) but consist of long chain amino alcohols forming amide bonds with fatty acid chains containing more than 18 carbons. PC and PE and glycosphingolipids may bind to CD1 molecules expressed by mammalian immune cells (e.g., Antigen Presenting Cells (APC), such as dendritic cells and macrophages) and other cells, including B and T lymphocytes in the thymus and liver. Furthermore, in addition to the potential adjuvant effect of the presence of plant lipids, the ability of plant N-glycans to facilitate capture of glycoprotein antigens by antigen presenting cells may also be advantageous for the production of chimeric VLPs in plants. Without wishing to be bound by theory, it is expected that chimeric VLPs produced by plants induce a stronger immune response than chimeric VLPs made in other production systems, and that chimeric VLPs produced by these plants induce a stronger immune response than that induced by live or attenuated whole virus vaccines.

Chimeric VLPs have advantages over vaccines made from whole viruses because they are non-infectious, and therefore the problem of restricted biocontainment is no longer as important as when using infectious whole viruses, and is not essential for production. Another advantage of chimeric VLPs produced by plants is that they allow the expression system to be grown in greenhouses or fields, thereby being significantly more economical and suitable for scale-up.

In addition, plants do not contain enzymes involved in the synthesis of sialic acid residues and their addition to proteins. VLPs may be produced without the need for Neuraminidase (NA), and without co-expressing NA or treating the producer cells or extracts with sialidase (neuraminidase) to ensure production of VLPs in plants.

This summary of the invention does not necessarily describe all inventive features of the invention.

Brief Description of Drawings

These and other features of the present invention will become more apparent from the following description with reference to the accompanying drawings, in which:

FIG. 1 shows a schematic of the HA subdomain. SP: signal peptides, F '1, F' 2 and F: a fusion subdomain; RB: receptor binding subdomains, E1 and E2: esterase subdomain, TMD/CT: transmembrane and cytoplasmic tail subdomains. FIG. 1B shows a schematic of plastocyanin-based expression cassettes (construct Nos. 774, 540, 660, 690, 691, 696) expressing hemagglutinin H1A/Brisban/59/2007 (H1/Bri), hemagglutinin H1A/neokaridonia/20/99 (H1/NC) and hemagglutinin H5A/Indonesian/5/05 (H5/Indo) in native and chimeric form. Plasto pro: the alfalfa plastocyanin promoter, Plasto ter: alfalfa plastocyanin terminator, SP: signal peptide, RB: receptor binding subdomain, E1-RB-E2: esterase and receptor binding subdomains, TMD/CT: transmembrane and cytoplasmic tail subdomain, PDI: alfalfa protein disulfide isomerase. Figure 1C shows an amino acid sequence alignment of several influenza HAs, with an additional structural alignment: (B/Florida/4/2006 (B Florida), SEQ ID NO: 94(GenBank accession ACA 33493.1; B/Malaysia/2506/2004 (B-Malaysia), SEQ ID NO: 95(GenBank accession ABU 99194.1); H1/Bri (A-Bri ban), SEQ ID NO: 96(GenBank accession ADE 28750.1; H1A/Sol island/3/2006 (A-Sol. isl), SEQ ID NO: 97(GenBank accession ABU 99109.1); H1/NC (A-NewCal), SEQ ID NO: 98(GenBank accession AAP 34324.1; H2A/Singapore/1/1957 (A-Singapore), SEQ ID NO: 99(GenBank accession AAA 63 64366.1); H3/Bri Banaban/10/2007 (A-Brix accession WCO A); SEQ ID NO: 68692/W accession ACA-100/W-K638); SEQ ID NO: 100 (ACH-Conn 638), SEQ ID NO: 101(GenBank accession No. ABO 37599.1); H5A/anhui/1/2005 (a-anhui), SEQ ID NO: 102(GenBank accession number ABD 28180.1); H5A/vietnam/1194/2004 (a-vietnam), SEQ ID NO: 103(GenBank accession number ACR 48874.1); H5-Indo, SEQ ID NO: 104(GenBank accession ABW06108.1. denotes F '1, esterase 1, receptor binding, esterase 2, F' 2, peptide fusion, the boundary between TMD/CT subdomains and disulfide bridges.

FIG. 2 shows the amino acid sequence of the subdomains shown by the chimeric HA expressed with constructs 690, 734(SEQ ID NO: 11), 696(SEQ ID NO: 112) (upper panel) and 691(SEQ ID NO: 113) (lower panel). SEQ ID NO: amino acids 1-92 of 111 are the F' 1+ E1 domain of H5/Indo; amino acids 93-263 are the RB head domain of H1/Brisbanl, and amino acids 264-552 are the E2+ F' 2 domain of H5/Indo. SEQ ID NO: amino acids 1-92 of 112 are the F' 1+ E1 domain of H5/NC; amino acids 93-301 are the RB head domain of H5/Indo, and amino acids 302-586 are the E2+ F' 2 domain of H1/NC. SEQ ID NO: amino acids 1-42 of 113 are the F' 1 domain of H5/Indo; amino acids 43-273 are the E1-RB-E2 head domain of H1/Brisbanl, and amino acids 274-552 are the F' 2 domain of H5/Indo.

FIG. 3 shows the amino acid sequence of the coding region of constructs 690 and 734(SEQ ID NO: 80) comprising the RB subdomain of H1/Bri, the H5/Indo signal peptide and a Stem Domain Complex (SDC) comprising H5/Indo F' 1, E1, E2 and the F subdomain.

FIG. 4 shows the amino acid sequence of the coding region of construct 691(SEQ ID NO: 81) comprising the H1/Bri Head Domain Complex (HDC) (comprising E1, RB, E2), the H5/Indo signal peptide and the Stem Domain Complex (SDC) comprising H5/Indo F '1, F' 2 and F subdomain.

FIG. 5 shows the amino acid sequence of the coding region of construct 696(SEQ ID NO: 82), which comprises the RB subdomain of H5/Indo, the PDI signal peptide, and the H1/NC stem domain complex comprising F '1, E1, E2, and F' 2.

FIG. 6 shows immunoblot analysis of H1/Bri expression in plants in native form, construct 774 (comprising H1/Bri), construct 692 (comprising the Head Domain Complex (HDC) of H1/Bri) and construct 690 (comprising the RB subdomain of H1/Bri fused to the H5/Indo Stem Domain Complex (SDC)). For each construct, total protein extracts from 3 independent plants were analyzed. The loading of protein was 20 micrograms per plant analyzed. Western blots were shown with anti-HA monoclonal antibodies (anti-H1-Brilliant Banner; FII 10-I50). Construct 774 expresses H1/Bri with the natural signal peptide of H1/Bri; constructs 690, 691 expressed HA with the H5/Indo native signal peptide.

FIG. 7 shows immunoblot analysis of H5/Indo expression in native form, construct 660 (containing H5/Indo) or construct 696 (containing H1/Indo RB subdomain fused to H1/NC SDC, E1 and E2 subdomain). For each construct, total protein extracts from 3 independent plants were analyzed. The loading of protein was 20 micrograms per plant analyzed. Western blotting was performed using an anti-H5 Indonesia polyclonal antibody (ITC IT-003-005V). Construct 660 expresses H5/Indo with its native signal peptide; construct 696 expresses chimeric HA with a PDI signal peptide.

FIG. 8 shows a schematic of a 35 SCPMV/HT-based expression cassette expressing both native (construct 732) and chimeric (constructs 733 and 734) forms of H1/Bri. Construct 733 contained the PDI signal peptide and H1/Bri's HDC, SDC, and Transmembrane Domain Complex (TDC), and construct 734 contained H5/Indo signal peptide, F ' 1, E1, E2, F ' 2, F, and RB from H1/Bri. 35S Dro: CaMV35S promoter, NOS ter: nopaline synthase terminator, SP: signal peptide, RB: receptor binding subdomain, E1-RB-E2: esterase and receptor binding subdomains, TMD/CT: transmembrane and cytoplasmic tail subdomain, PDI: alfalfa protein disulfide isomerase; CPMV-HT: the 5 'and 3' elements of the over-translatable (hyper translatable) cowpea mosaic virus expression system.

FIG. 9 shows an immunoblot analysis of H1/Bri expression in native form, construct 732 (comprising H1/Bri under the control of a 35 SCPMV/HT-based expression cassette), construct 733 (comprising a PDI signal peptide fused to H1/Bri; under the control of a 35 SCPMV/HT-based expression cassette) or construct 734 (comprising H1/Bri RB subdomain fused to H5/IndosDC, E1 and E2 subdomains; under the control of a 35 SCPMV/HT-based expression cassette). For each construct, total protein extracts from 3 independent plants were analyzed. The loading protein was 5 μ g for each plant analyzed. Western blots were revealed by detection with anti-HA monoclonal antibodies (FII 10-I50).

FIG. 10 shows a schematic of 35 SCPMV/HT-based expression cassettes expressing H3A/Brilliant/10/2007 (H3/Bri) and B/Florida/4/2006 HA (B/Flo) hemagglutinin. Construct 736 contained H3/Bri fused to the PDI signal peptide. Construct 737 contained H3/Bri and H5/Indo TMD/CT fused to the PDI signal peptide. Construct 739 contained B/Flo fused to PDI signal peptide. Construct 745 contained B/Flo and H5/IndoTMD/CT fused to the PDI signal peptide. 35S pro: CaMV35S promoter, NOS ter: nopaline synthase terminator, SP: signal peptide, RB: receptor binding subdomain, E1-RB-E2: esterase and receptor binding subdomains, TMD/CT: transmembrane and cytoplasmic tail subdomain, PDI: alfalfa protein disulfide isomerase; CPMV-HT: 5 'and 3' elements of an over-translatable cowpea mosaic virus expression system.

Fig. 11 shows the fusion boundaries in constructs 745 and 737. The start of the HA sequence is indicated by the cone headed arrow. The transmembrane domain has amino acid QILSIYSTVA, preceded by a partial extracellular domain of amino acids.

FIG. 12 shows the amino acid sequence of chimeric H5/H3 hemagglutinin (SEQ ID NO: 83, construct 737) comprising the PDI signal peptide, the extracellular domain of H3A/Brisbane/10/2007, and TMD/CT of H5A/Indonesia/5/2005.

FIG. 13 shows the amino acid sequence of chimeric H5/B hemagglutinin (SEQ ID NO: 84), which comprises the extracellular domain of B/Florida/4/2006 encoded by the open reading frame of construct No. 745 and TMD/CT of H5A/Indonesia/5/2005.

FIG. 14 shows immunoblot analysis of B/Flo expression in native form, construct 739 (comprising PDI-B/Flo) or construct 745 (comprising B/Flo HDC and SDC fused to H5/Indo TDC). For each construct, total protein extracts from 3 independent plants were analyzed. The loading of protein was 20 micrograms per plant analyzed. Western blots were shown with anti-HA B/Florida polyclonal antibody (NIBSC 07/356).

FIG. 15 shows immunoblot analysis of H3/Bri expression of native form, construct 736 (comprising PDI sp-H3/Bri) or construct 737 (H3/Bri HDC and SDC fused to H5/Indo TDC). For each construct, total protein extracts from 3 independent plants were analyzed. The loading of protein was 20 micrograms per plant analyzed. Western blots were shown with anti-H3 Brisban polyclonal antibody (NIBSC 08/124).

Figure 16 shows size exclusion chromatography of leaf protein extracts of plants infected with construct No. 745. For each fraction, the relative protein content of the eluted fractions is shown. Immunodetection (Western blot) of hemagglutinin in fractions 7 to 15 using anti-HA B/Florida polyclonal antibody (NIBSC 07/356) is shown below. The arrow indicates the elution peak of blue dextran 2000 (fraction 8).

FIG. 17 shows the nucleic acid sequence of the synthetic fragment (SEQ ID NO: 52) comprising the entire H5 (A/Indonesia/5/05 (H5N1)) coding region (containing the signal peptide and stop codon), flanked at 5 'by HindIII sites and at 3' by SacI sites.

FIG. 18 shows the nucleic acid sequence (SEQ ID NO: 53) of construct 660, construct 660 being an HA expression cassette comprising the alfalfa plastocyanin promoter and 5 'UTR, the coding sequence for type H5A/Indonesia/5/05 (H5N1) hemagglutinin, alfalfa plastocyanin 3' UTR and terminator sequences.

FIG. 19 shows the nucleic acid sequence (SEQ ID NO: 54) of the coding sequence of wild-type H1 (A/New Cardonia/20/99 (H1N1)) (GenBank accession AY289929) without TmD and Ctail.

FIG. 20 shows the nucleic acid sequence (SEQ ID NO: 55) of a synthetic fragment containing the H1 (A/New Caledonia/20/99 (H1N1)) coding sequence lacking TmD and Ctail. In the 5 'region, the last few nucleotides are from PDI SP and contain a BglII restriction site, located immediately downstream of the stop codon at the 3' SacI/StuI dibit site.

FIG. 21 shows the nucleic acid sequence (SEQ ID NO: 56) of a synthetic fragment containing the C-ter H1 (A/New Callidonia/20/99 (H1N1)) coding sequence (containing TmD and Ctail) from the KpnI site to the stop codon (flanked 3' by SacI/StuI double sites).

FIG. 22 shows the nucleotide sequence of protein disulfide isomerase mRNA from Medicago sativa (Medicago sativa). GenBank accession number Z11499(SEQ ID NO: 57). Nucleotides 32-103 encode the PDI signal peptide.

FIG. 23 shows the nucleotide sequence of the PromPlasto-PDISP-Plasto 3' UTR plasmid. FIG. 23A shows the nucleotide sequence of PromPlasto-PDISP (SEQ ID NO: 58). FIG. 23B shows the nucleotide sequence of the Plasto 3' UTR (SEQ ID NO: 85). The Protein Disulfide Isomerase (PDI) signal peptide sequence is underlined. BglII (AGATCT) and SacI (GAGCTC) restriction sites used for cloning are shown in bold.

FIG. 24 shows the nucleic acid sequence of an HA expression cassette (SEQ ID NO: 59; construct 540) comprising the alfalfa plastocyanin promoter and 5 'UTR, PDI signal peptide and the coding sequence for type H1A/New Cardonia/20/99 (H1N1), alfalfa plastocyanin 3' UTR and terminator sequences. The H1 coding sequence from A/New Caledonia/20/1999 is underlined.

FIG. 25 shows the nucleic acid sequence of the synthetic fragment (SEQ ID NO: 60) comprising the entire H1 (A/Brisbane/59/07 (H1N1)) coding region (containing the signal peptide and stop codon), flanked 5 'by the sequence of the alfalfa plastocyanin gene starting with a DraIII site (corresponding to the first 84 nucleotides upstream of the starting ATG), and 3' by a SacI site.

FIG. 26 shows the nucleic acid sequence of an HA expression cassette (SEQ ID NO: 61, construct 774) comprising the alfalfa plastocyanin promoter and 5 'UTR, hemagglutinin coding sequence of type H1A/Brilliban/59/07 (H1N1), alfalfa plastocyanin 3' UTR, and terminator sequences.

FIG. 27 shows the nucleic acid sequence of expression cassette number 828(SEQ ID NO: 62), from PacI (upstream of the promoter) to AscI (immediately downstream of the NOS terminator). The CPMV HT 3' UTR sequence is underlined, with the mutated ATG in bold. The Apa1 restriction site is underlined and italicized.

FIG. 28 shows the nucleic acid sequence of a chimeric H5/H1 expression cassette (SEQ ID NO: 63, construct 690) comprising an alfalfa plastocyanin promoter and 5 'UTR, a chimeric hemagglutinin coding sequence, alfalfa plastocyanin 3' UTR, and a terminator sequence. The chimeric HA coding sequence is underlined.

FIG. 29 shows the nucleic acid sequence of a chimeric H5/H1 expression cassette (SEQ ID NO: 64, construct 691) comprising an alfalfa plastocyanin promoter and 5 'UTR, a chimeric hemagglutinin coding sequence, alfalfa plastocyanin 3' UTR, and a terminator sequence. The chimeric HA coding sequence is underlined.

FIG. 30 shows the nucleic acid sequence of a chimeric H1/H5 expression cassette (SEQ ID NO: 65, construct 696) comprising an alfalfa plastocyanin promoter and 5 'UTR, a chimeric hemagglutinin coding sequence, alfalfa plastocyanin 3' UTR, and a terminator sequence. The chimeric HA coding sequence is underlined.

FIG. 31 shows the nucleic acid sequence of the HA expression cassette (SEQ ID NO: 66, construct 732) comprising the CaMV35S promoter, CPMV-HT5 'UTR, type H1A/Brilliban/59/07 (H1N1) hemagglutinin coding sequence, CPMV-HT 3' UTR and NOS terminator sequences. The coding sequence of H1/Bri is underlined.

FIG. 32 shows the nucleic acid sequence of intermediate construct 787 from ATG to the coding sequence of termination (SEQ ID NO: 67).

FIG. 33 shows the nucleic acid sequence of the SpPDI H1/Bri expression cassette (SEQ ID NO: 68, construct No. 733) comprising the CaMV35S promoter, CPMV-HT5 'UTR, PDI signal peptide coding sequence, H1 type A/Brilliban/59/07 (H1N1) hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences. The SpPDI H1/Bri coding sequence is underlined.

FIG. 34 shows the nucleic acid sequence of a chimeric H5/H1 expression cassette (SEQ ID NO: 69, construct 734) comprising a CaMV35S promoter, a CPMV-HT5 'UTR, a chimeric hemagglutinin coding sequence, a CPMV-HT 3' UTR, and a NOS terminator sequence. The coding sequence of the chimeric HA is underlined.

FIG. 35 shows the nucleic acid sequence (SEQ ID NO: 70) of a synthetic fragment comprising the entire H3 (A/Brisbane/10/07 (H3N2)) coding region (containing the signal peptide and stop codon), 5 'flanked by the sequence of the alfalfa plastocyanin gene starting with a DraIII site (corresponding to the first 84 nucleotides upstream of the starting ATG), and 3' flanked by a SacI site.

FIG. 36 shows the nucleic acid sequence of an HA expression cassette comprising the CaMV35S promoter, CPMV-HT5 'UTR, the PDI signal peptide coding sequence, the H3 type A/Brilliant Band/10/07 (H2N3) hemagglutinin coding sequence, CPMV-HT 3' UTR, and the NOS terminator sequence (SEQ ID NO: 71, construct 736). The Sp PDI H3/Bris coding sequence is underlined.

FIG. 37 shows the nucleic acid sequence (SEQ ID NO: 72, construct No. 737) of the chimeric H5/H3 expression cassette comprising the CaMV35S promoter, CPMV-HT5 'UTR, chimeric hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences. The coding sequence of the chimeric HA is underlined.

FIG. 38 shows the nucleic acid sequence (SEQ ID NO: 73) of a synthetic fragment containing the entire HA (B/Florida/4/06) coding region (containing the signal peptide and stop codon), 5 'flanked by the sequence of the lucerne plastocyanin gene starting with the DraIII site (corresponding to the first 84 nucleotides upstream of the starting ATG), and 3' flanked by the SacI site.

FIG. 39 shows the nucleic acid sequence of an HA expression cassette comprising the CaMV35S promoter, CPMV-HT5 'UTR, PDI signal peptide coding sequence, HA type B/Florida/4/06 hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences (SEQ ID NO: 74, construct 739). The Sp PDI B/Flo coding sequence is underlined.

FIG. 40 shows the nucleic acid sequence of a chimeric H5/B expression cassette (SEQ ID NO: 75, construct 745) comprising a CaMV35S promoter, CPMV-HT5 'UTR, a chimeric hemagglutinin coding sequence, CPMV-HT 3' UTR, and a NOS terminator sequence. The coding sequence of the chimeric HA is underlined.

FIG. 41 shows the nucleic acid sequence encoding Msj 1(SEQ ID NO: 76).

FIG. 42 shows the nucleic acid sequence of part of construct number R850(SEQ ID NO: 77), from HindIII (in the multiple cloning site, upstream of the promoter) to EcoRI (immediately downstream of the NOS terminator). The HSP40 coding sequence is underlined.

FIG. 43 shows the nucleic acid sequence of part of construct number R860(SEQ ID NO: 78), from HindIII (in the multiple cloning site, upstream of the promoter) to EcoRI (immediately downstream of the NOS terminator). The HSP70 coding sequence is underlined.

FIG. 44 shows the nucleic acid sequence of part of construct number R870 (SEQ ID NO: 79), from HindIII (5' upstream of the promoter in the multiple cloning site) to EcoRI (immediately downstream of the NOS terminator). The HSP40 coding sequence is underlined and italicized and the HSP70 coding sequence is underlined. A) Nucleotides 1 to 4946; B) nucleotides 4947 and 9493.

Figure 45 shows a schematic of construct R472.

Figure 46 shows disulfide bridge pattern of influenza a. Bridge numbering: 1) cys4HA1-Cys137HA2, 2) Cys60HA1-Cys72HA1, 3) Cys94HA1-Cys143HA1, 4) Cys292HA1-Cys318HA1, 5) Cys144HA2-Cys148HA2, and 6) Cys52HA1-Cys277HA 1. The difference in disulfide bridges between the A and B subtypes (FIG. 47) is indicated by arrows. The numbering of the mature H3 protein was used.

Figure 47 shows the disulfide bridge pattern of influenza b HA. Bridge numbering: 1) cys4HA1-Cys137HA2, 2) Cys60HA1-Cys72HA1, 3) Cys94HA1-Cys143HA1, 4) Cys292HA1-Cys318HA1, 5) Cys144HA2-Cys148HA2, 6) Cys52HA1-Cys277HA1, 7) Cys54HA1-Cys57HA1, and 8) Cys178HA1-Cys272HA 1. The difference in disulfide bridges between the A and B subtypes (FIG. 46) is indicated by arrows. The numbering of the mature H3 protein was used.

FIG. 48 shows a schematic of domain replacement (swap) fusion junctions. FIG. 48A shows the fusion of RB subdomains from H1/Bri, H3/Bri and B/Flo with H5/Indo SDC and the fusion of the RB subdomain of H5/Indo with H1/NC stem domain. FIG. 48B shows the fusion of E1-RB-E2 subdomain (HDC) from H1/Bri, H3/Bri or B/Flo with H5/Indo SDC, and H5/Indo HDC with H1/NC SDC.

FIG. 49A shows the nucleotide sequence of H1A/California/04/09 (SEQ ID NO: 86). The alfalfa protein disulfide isomerase signal peptide coding sequence is underlined and the mature H1 coding sequence is highlighted in bold. FIG. 49B shows the amino acid sequence of H1A/California/04/09 (SEQ ID NO: 87). The alfalfa protein disulfide isomerase signal peptide sequence is underlined.

FIG. 50 shows an immunoblot analysis of the expression of H5/B chimeric hemagglutinin (construct 747, comprising B/Flo HDC and SDC fused to H5/Indo TDC) following infection with undiluted AGL1/747, co-infection with AGL1/443 (empty vector) and co-infection with AGL1/R870(HSP40/HSP 70). For each construct, total protein extracts from 3 independent plants were analyzed. The loading of protein was 20 micrograms per plant analyzed. Western blots were shown with anti-B/Florida polyclonal antibody (NIBSC).

FIG. 51A shows the nucleotide sequence of the 2X35S promoter sequence (SEQ ID NO: 88). FIG. 51B shows the nucleotide sequence of construct 747(SEQ ID NO: 93) from PacI (upstream of the 35S promoter) to AscI (immediately downstream of the NOS terminator). The coding sequence of the chimeric HA is underlined. The 2X35S promoter sequence is in italics.

Detailed Description

A preferred embodiment is described below.

The present invention provides nucleic acids comprising a nucleotide sequence encoding a chimeric influenza Hemagglutinin (HA) operably linked to a regulatory region active in plants.

Furthermore, the present invention provides a method of producing Virus Like Particles (VLPs) in a plant. The method comprises introducing into a plant or plant part a nucleic acid sequence encoding a chimeric influenza HA (operably linked to a regulatory region active in the plant) and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The invention also provides VLPs comprising chimeric influenza HA. VLPs can be produced by the methods provided by the invention.

"chimeric protein" or "chimeric polypeptide" refers to a protein or polypeptide comprising amino acid sequences from two or more sources, such as, but not limited to, two or more influenza types or subtypes fused as a single polypeptide or influenza of different origin. The chimeric protein or polypeptide may comprise a signal peptide that is the same or heterologous to the remaining polypeptide or protein. The chimeric protein or chimeric polypeptide can be transcribed as a transcript from the chimeric nucleotide sequence, the chimeric protein or chimeric polypeptide is cleaved after synthesis, and if desired, combined to form a multimeric protein. Thus, chimeric proteins or chimeric polypeptides also include proteins or polypeptides comprising subunits linked by disulfide bridges (i.e., multimeric proteins). For example, a chimeric polypeptide comprising amino acid sequences from two or more sources can be processed into subunits that are joined by disulfide bridges to produce a chimeric protein or chimeric polypeptide (see fig. 46 and 47). The polypeptide may be Hemagglutinin (HA), and the two or more amino acid sequences each forming the polypeptide may be obtained from different HAs to produce a chimeric HA or a chimeric influenza HA. Chimeric HA may also include amino acid sequences comprising a heterologous signal peptide (chimeric HA precursor protein) that is cleaved after or during protein synthesis. Preferably, the chimeric polypeptide or chimeric influenza HA is not native. A nucleic acid encoding a chimeric polypeptide may be described as a "chimeric nucleic acid" or "chimeric nucleotide sequence". Virus-like particles comprising chimeric HA may be described as "chimeric VLPs".

The chimeric influenza HA of various embodiments of the invention may comprise a Stem Domain Complex (SDC), a Head Domain Complex (HDC), and a Transmembrane Domain Complex (TDC), wherein one or more subdomains of the SDC, HDC, or TDC are from a first influenza HA type, subtype, or from one source, and one or more subdomains of the SDC, HDC, or TDC are from a second influenza HA type, subtype, or from a second or different source. As described herein, "SDC" comprises F '1, F' 2 and F subdomains, "HDC" comprises RB, E1 and E2 subdomains, and "TDC" comprises TmD and Ctail subdomains (TMD/CT; see FIGS. 1A, 46 and 47).

The term "virus-like particle" (VLP) or "VLP" refers to a structure that is self-assembling and comprises a structural protein, such as an influenza HA protein or a chimeric influenza HA protein. VLPs and chimeric VLPs are generally similar in morphology and antigenicity to virions produced in infection, but lack sufficient genetic information to replicate and are therefore not infectious. VLPs and chimeric VLPs may be produced in suitable host cells, including plant host cells. After extraction from host cells, separation under appropriate conditions and further purification, VLPs and chimeric VLPs can be purified as intact structures.

The chimeric VLP or VLP of the present invention produced from an influenza-derived protein does not comprise M1 protein. The M1 protein is known to bind RNA (Wakefield and Brown, 1989), which is a contaminant in the preparation of VLPs. The presence of RNA is undesirable when the chimeric VLP product is subject to regulatory approval, and therefore a chimeric VLP formulation lacking RNA is advantageous.

Chimeric VLPs of the invention may be produced in host cells characterized by a lack of the ability to sialylate proteins, such as plant cells, insect cells, fungi and other organisms (including sponges, coelenterates, annelids, arthropods, molluscs, lineages (nemathelminthhemantinheas), trochelxints, platoons, chinchillas, tentacles, chlamydia, spirochetes, gram positive bacteria, cyanobacteria, archaebacteria, and the like. see, e.g., Gupta et al, 1999.Nucleic Acids Research 27: 370-372; Toukach et al, 2007.Nucleic Acids Research 35: D280-D286; Nakahara et al, 2008.Nucleic Acids Research 36: D368-D371. VLPs produced as described herein typically do not contain Neuraminidase (NA). however, if it is desired to obtain VLPs comprising HA and NA, the NA can be co-expressed with HA.

The invention also provides VLPs comprising chimeric HA having a lipid envelope obtained from the plasma membrane of cells expressing the chimeric HA. For example, if the chimeric HA is expressed in a plant-based system, the resulting VLP may obtain a lipid envelope from the plasma membrane of the plant cell.

In general, the term "lipid" refers to a fat-soluble (lipophilic) natural molecule. Chimeric VLPs produced in plants according to some aspects of the invention may form complexes with plant-derived lipids. The plant-derived lipid may be in the form of a lipid bilayer and may also comprise an envelope that encapsulates the VLP. The plant-derived lipid may comprise plasma membrane lipid components of the plant producing the VLPs, including phospholipids, tri-, di-and monoglycerides, and lipid-soluble sterols or metabolites comprising sterols. Examples include Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), phosphatidylinositol, phosphatidylserine, glycosphingolipids, phytosterols, or combinations thereof. Plant-derived lipids may also be referred to as "plant lipids". Examples of phytosterols include campesterol, stigmasterol, ergosterol, brassicasterol, Δ -7-stigmasterol, Δ -7-avenasterol, daunosterol, sitosterol, 24-methylcholesterol, cholesterol, or β -sitosterol-see, for example, Mongrand et al, 2004. It will be appreciated by those skilled in the art that the lipid composition of the plasma membrane of a cell may vary depending on the culture or growth conditions of the cell or organism or species from which the cell is obtained. In general, β -sitosterol is the most abundant phytosterol.

The cell membrane typically comprises a lipid bilayer as well as multifunctional proteins. A localized concentration of specific lipids, called "lipid rafts", can be found in the lipid bilayer. Sphingolipids and sterols are rich in these lipid raft microdomains. Without wishing to be bound by theory, lipid rafts may play an important role in endocytosis and exocytosis, entry or egress of viruses or other infectious agents, intercellular signal transduction, interactions with other structural components of cells or organisms (e.g., intracellular and extracellular matrices).

The present invention includes VLPs comprising chimeric HA, wherein the subdomain of HA may be obtained from any type, subtype of influenza virus that can infect humans, including, for example, B, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 types or subtypes. In some embodiments, the influenza virus may be H1, H3, H5, or a type B or subtype. Non-limiting examples of types or subtypes of H1, H3, H5 or B include type A/Neocarinidonia/20/99 subtype (H1N1) ("H1/NC"; SEQ ID NO: 56), H1A/California/04/09 subtype (H1N1) ("H1/Cal"; SEQ ID NO: 86), type A/Indonesian/5/05 (H5N1) ("H5/Indo"), A/Brillian/59/2007 ("H1/Bri"), and B/Florida/4/2006 ("B/Flo") and H3A/Brillian/10/2007 ("H3/Bri"). Also, the chimeric HA may comprise one or more hemagglutinin subdomains isolated from one or more existing or newly identified influenza viruses.

The invention also relates to influenza viruses that infect other mammals or host animals, such as humans, primates, horses, pigs, birds, waterfowls, migratory birds, quails, ducks, geese, poultry, chickens, camels, canines, dogs, felines, cats, tigers, leopards, muskrat, mink, ferrets, pets, livestock, mice, rats, seals, whales, and the like. Some influenza viruses can infect more than one host animal.

For influenza viruses, the term "hemagglutinin" or "HA" as used herein refers to the structural glycoprotein of the influenza virion. The structure of influenza hemagglutinin has been studied extensively and shows highly conserved secondary, tertiary and quaternary structures. This structural conservation is observed even if the amino acid sequence is changed (see, e.g., Skehel and Wiley, 2000 Ann Rev Biochem 69: 531-69; Vaccaro et al 2005, incorporated herein by reference). Nucleotide sequences encoding HA are well known and available, see, e.g., biodefenses and Public Health base (e.g., URL: biohealth base. org/GSearch/home. dococorater. infiluenza) or the american center for biotechnology information (NCBI; see URL: NCBI. nlm. nih. gov/sites/enterzzdb. nuccrere & cm. search & term), both of which are incorporated herein by reference.

The HA monomer can be further divided into 3 functional domains-stem domain or cluster of Stem Domains (SDC), globular head domain or cluster of Head Domains (HDC) and cluster of Transmembrane Domains (TDC). SDC comprises 4 subdomains, fusion peptides F, F '1 and F' 2 (the subdomains may be generally referred to as "backbones"). TDC comprises two subdomains, transmembrane (TmD) and C-terminal tail (CT). HDC comprises three subdomains, the residual esterase domains E1' and E2 and the receptor binding domain RB. SDC and HDC may be collectively referred to as "extracellular domains. Ha et al 2002(EMBO J.21: 865-. FIG. 1A shows a schematic representation of subdomains associated with the N-and C-termini of HA1 and HA2 polypeptides. Figure 1C provides a labeled structural alignment of multiple influenza subtypes.

Amino acid changes are allowed in influenza virus hemagglutinin. This change provides new strains that are continually being identified. Infectivity may vary between new strains. However, the formation of hemagglutinin trimers, which subsequently form VLPs, is maintained. The invention thus provides nucleic acids comprising the hemagglutinin amino acid sequence of a chimeric HA or encoding a chimeric hemagglutinin amino acid sequence that forms VLPs in plants, and includes known sequences and variant HA sequences that can be produced. The invention also relates to the use of a chimeric HA polypeptide comprising TDC, SDC and HDC. For example, the chimeric HA protein may be HA0, or a cleaved chimeric HA comprising HA1 and HA2 subdomains from two or more influenza types. The chimeric HA proteins may be used to produce or form VLPs using plants or plant cell expression systems.

HA0 may be expressed and folded to form trimers, which may then be assembled into VLPs. Cleavage of HA0 produced HA1 and HA2 polypeptides, which were linked by disulfide bridges (see fig. 1C, 46 and 47 for a graphical representation of the disulfide bridge pattern). For infectious viral particles, cleavage of the precursor HA0 is required to initiate a conformational change in HA2 which releases the fusion peptide (at the N-terminus of the HA2 polypeptide) and makes it available for fusion with cellular and viral membranes. However, VLPs are not infectious and do not require cleavage of HA to form HA1 and HA2 (e.g. in vaccine production). Uncleaved HA0 precursors also assemble into trimers and bud from the plasma membrane to form VLP nanoparticles.

The HA0 polypeptide comprises several domains. The RB subdomain of HDC contains several loops in the antigenic region, designated sites A-E. Antibodies that can neutralize infectious influenza viruses often target one or more of these sites. The remnant esterase subdomains (E1 and E2) may function in fusion and may bind Ca + +. F. The F '1 and F' 2 domains interact and cooperate to form a stem, such that the head of the HA trimer is located above the membrane. TmD and CT may participate in the anchoring of folded HA to the membrane. TmD may play a role in the affinity of HA for lipid rafts, whereas CT may play a role in the secretion of HA, and some of the cysteine residues present in the CT subdomain may be palmitoylated. A Signal Peptide (SP) may also be present at the N-terminus of the HA0 polypeptide. Fig. 2 and tables 4 and 5 provide examples of amino acid sequences of the SP, F '1, F' 2, E1, RB, E2, and F domains of some influenza virus subtypes.

Processing of the N-terminal Signal Peptide (SP) sequence during expression and/or secretion of influenza hemagglutinin may play a role in HA folding. The term "signal peptide" generally refers to a short (about 5-30 amino acids) amino acid sequence, typically found at the N-terminus of a hemagglutinin polypeptide, that can direct translocation of a newly translated polypeptide to a particular organelle, or aid in the localization of a particular domain of a polypeptide chain relative to other domains. The signal peptide of hemagglutinin targets translocation of the protein to the endoplasmic reticulum, which has been proposed to aid in the localization of the proximal N-domain relative to the membrane anchoring domain of nascent hemagglutinin polypeptides to aid in cleavage and folding of mature hemagglutinin.

Insertion of HA within the Endoplasmic Reticulum (ER) membrane of the host cell, signal peptide cleavage, and protein glycosylation are cotranslational events. Proper folding of HA requires protein glycosylation and formation of at least 6 intrachain disulfide bonds (see fig. 46 and 47). In fig. 46, each monomer of subtype a HA is shown to have 6 conserved disulfide bridges. By comparison, the monomers of B HA (FIG. 47) have 7 disulfide bridges, 5 of which have corresponding structures in type A (reviewed in Skehel and Wiley, 2000.Ann RevBiochem 69: 531-569; structural examples illustrating intramolecular and intermolecular disulfide bridges and other conserved amino acids and their relative positions are described, e.g., in Gamblin et al 2004, Science 303: 1838-1842, both of which are incorporated herein by reference). It will be appreciated by those skilled in the art that it is important to prepare chimeric HA to ensure that similarly aligned disulfide bridges are obtained.

The signal peptide may be naturally occurring in hemagglutinin, or the signal peptide may be heterologous to the primary sequence from which hemagglutinin is expressed. The chimeric HA may comprise a signal peptide from a first influenza type, subtype or strain, with the remaining HA from one or more different influenza types, subtypes or strains. Natural SPs such as HA subtypes B H1, H2, H3, H5, H6, H7, H9 or influenza b can be used to express HA in plant systems. In some embodiments of the invention, the SP may be from influenza b, H1, H3, or H5; or from subtype H1/Bri, H1/NC, H5/Indo, H3/Bri or B/Flo.

The SP may also be non-native, such as from a structural protein or hemagglutinin of a virus other than an influenza virus, or from a plant, animal or bacterial polypeptide. A non-limiting example of a signal peptide that may be used is alfalfa protein disulfide isomerase (PDI SP; nucleotides 32-103 of accession number Z11499; SEQ ID NO: 34; FIG. 17) having the amino acid sequence:

MAKNVAIFGLLFSLLLLVPSQIFAEE (nucleotides 32-103; SEQ ID NO: 34).

The invention thus provides chimeric influenza hemagglutinin comprising a native or non-native signal peptide and nucleic acids encoding the chimeric hemagglutinin.

Proper folding of hemagglutinin can be important for influenza hemagglutinin properties such as protein stability, multimer formation, VLP formation, and HA function (hemagglutination capacity). Protein folding may be affected by one or more factors, including but not limited to: protein sequence, relative abundance of the protein, degree of intracellular crowding, availability of cofactors that can bind or transiently associate with a folded, partially folded or unfolded protein, presence of one or more chaperones, and the like.

Heat shock proteins (Hsp) or stress proteins are examples of chaperones that can be involved in a variety of cellular processes, including protein synthesis, intracellular trafficking, prevention of misfolding, prevention of protein aggregation, assembly and disassembly of protein complexes, protein folding, and protein disaggregation. Examples of such chaperones include, but are not limited to, Hsp60, Hsp65, Hsp70, Hsp90, Hsp100, Hsp20-30, Hsp10, Hsp100-200, Hsp100, Hsp90, Lon, TF55, FKBP, cyclophilin (cyclophilin), ClpP, GrpE, ubiquitin, calnexin (calnexin), and protein disulfide isomerase (see, e.g., Macario, A.J.L., Cold Spring harbor laboratory Res.25: 59-70.1995; Parsell, D.A. & Lindqquist, S.Ann.Rev.Genet.27: 437-496 (1993); U.S. Pat. No.5,232,833). As described herein, chaperonins (such as, but not limited to Hsp40 and Hsp70) may be used to ensure folding of the chimeric HA.

Examples of Hsp70 include Hsp72 and Hsc73 from mammalian cells, DnaK from bacteria, particularly mycobacteria such as Mycobacterium leprae, Mycobacterium tuberculosis and Mycobacterium bovis (e.g. Bacillus Calmette Guerin: herein referred to as Hsp 71). DnaK from E.coli, yeasts and other prokaryotes, and BiP and Grp78 from eukaryotes, such as Arabidopsis (A. thaliana) (Lin et al, 2001(Cell Stress and C photons 6: 201-208)). A specific example of Hsp70 is Arabidopsis Hsp70 (encoded by Genbankref: AY 120747.1). Hsp70 is capable of specifically binding ATP as well as unfolded polypeptides and peptides, thereby participating in protein folding and unfolding and assembly and disassembly of protein complexes.

Examples of Hsp40 include DnaJ from prokaryotes (e.g.E.coli and Mycobacteria) and HSJ1, HDJ1 and Hsp40 from eukaryotes (e.g.alfalfa) (Frugis et al, 1999 Plant Molecular Biology 40: 397-408). A specific example of Hsp40 is alfalfa (M.sativa) MsJ1(AJ000995.1 or SEQ ID NO: 76). Hsp40 acts as a chaperone in cellular activities such as protein folding, thermotolerance and DNA replication. FIG. 41 shows the nucleic acid sequence encoding Msj 1(SEQ ID NO: 76).

In Hsp, Hsp70 and its co-chaperone (co-chaperone) Hsp40 are involved in the stabilization of both the now-translated and newly synthesized polypeptides prior to completion of synthesis. Without wishing to be bound by theory, Hsp40 binds to the hydrophobic patch (patch) of an unfolded (nascent or newly transferred) polypeptide, thereby facilitating the interaction of the Hsp70-ATP complex with the polypeptide. ATP hydrolysis leads to the formation of a stable complex between the polypeptide, Hsp70 and ADP and the release of Hsp 40. The association of the Hsp70-ADP complex with the hydrophobic domain of the polypeptide prevents its interaction with other hydrophobic domains, preventing incorrect folding and aggregation with other proteins (reviewed in Hartl, FU.1996.Nature 381: 571-579).

Native chaperones may be able to facilitate proper folding of low levels of recombinant protein, however as expression levels increase, the abundance of native chaperones may become a limiting factor. High levels of expression of hemagglutinin in agrobacterium-infected leaves can result in accumulation of hemagglutinin polypeptides in the cytosol, while co-expression of one or more chaperones (e.g., Hsp70, Hsp40, or both Hsp70 and Hsp 40) can reduce the level of misfolded or aggregated hemagglutinin polypeptides and increase the number of polypeptides that exhibit tertiary and quaternary structural properties that allow hemagglutination and/or virus-like particle formation. SEQ ID NO: 77 is the partial nucleic acid sequence of construct number R850 from HindIII (upstream of the promoter in the multiple cloning site) to EcoRI (immediately downstream of the NOS terminator), which encodes HSP40 (underlined). SEQ ID NO: 78 is the partial nucleic acid sequence of construct No. R860 from HindIII (in the multiple cloning site, upstream of the promoter) to EcoRI (immediately downstream of the NOS terminator), which encodes HSP70 (underlined). SEQ ID NO: 79 is the partial nucleic acid sequence of construct number R870 from HindIII (5' upstream of the promoter in the multiple cloning site) to EcoRI (immediately downstream of the NOS terminator) which encodes HSP40 (underlined, italicized) and HSP70 (underlined).

Accordingly, the present invention also provides a method of producing chimeric influenza VLPs in a plant, wherein a first nucleic acid encoding a chimeric influenza HA is co-expressed with a second nucleic acid encoding a chaperone protein. The first and second nucleic acids may be introduced into the plant in the same step, or may be introduced into the plant sequentially.

The structure and size of VLPs can be assessed by, for example, hemagglutination assays, electron microscopy observations or size exclusion chromatography.

For size exclusion chromatography, all soluble proteins can be extracted from plant tissues by: homogenizing frozen and pulverized plant material in extraction buffer (Polytr)on), insoluble material was removed by centrifugation. Precipitation with PEG may also be advantageous. Quantifying the soluble protein and passing the extract through Sephacryl^TMAnd (3) a column. Blue dextran 2000 can be used as a calibration standard. After chromatography, the fractions may be further analyzed by immunoblotting to determine the protein content of the fractions.

The invention also provides plants comprising nucleic acids encoding one or more chimeric influenza hemagglutinin and nucleic acids encoding one or more chaperone proteins.

The invention comprises the nucleotide sequence:

SEQ ID NO: 63 (construct 690; chimeric H5/H1 expression cassette comprising an alfalfa plastocyanin promoter and 5 'UTR, chimeric hemagglutinin coding sequence, alfalfa plastocyanin 3' UTR and terminator sequences), SEQ ID NO: 63 underlined parts encode SP, F '1 of H5/Indo, E2, F' 2, F, TMD/CT of RB-H5/Indo of E1-H1/Bri;

SEQ ID NO: 64 (construct 691; chimeric H5/H1 expression cassette comprising an alfalfa plastocyanin promoter and 5 'UTR, chimeric hemagglutinin coding sequence, alfalfa plastocyanin 3' UTR, and terminator sequences), SEQ ID NO: the 64 underlined parts encode SP of H5/Indo, E1 of F '1-H1/Bri, RB, F' 2 of E2-H5/Indo, F, TMD/CT;

SEQ ID NO: 65 (construct 696; chimeric H1/H5 expression cassette comprising an alfalfa plastocyanin promoter and 5 'UTR, chimeric hemagglutinin coding sequence, alfalfa plastocyanin 3' UTR, and terminator sequences), SEQ ID NO: 65 underlined parts encode F '1 of PDI SP-H1/NC, E2, F' 2, F, TMD/CT of RB-H1/NC of E1-H5/Indo;

SEQ ID NO: 68 (construct 733; SpPDI H1/Bri expression cassette comprising CaMV35S promoter, CPMV-HT5 'UTR, PDI signal peptide coding sequence, H1 type a/brisbane/59/07 (H1N1) hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences), SEQ ID NO: the underlined portion of 68 encodes F '1, E1, RB, E2, F' 2, F, TMD/CT of PDI SP-H1/BRI;

SEQ ID NO: 69 (construct 734; chimeric H5/H1 expression cassette comprising CaMV35S promoter, CPMV-HT5 'UTR, chimeric hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences). The coding sequence of the chimeric HA is underlined, which encodes a sequence identical to seq id NO: 63 identical chimeric HA;

SEQ ID NO: 71 (construct 736; HA expression cassette comprising CaMV35S promoter, CPMV-HT5 'UTR, PDI signal peptide coding sequence, H3 type a/brisbane/10/07 (H2N3) hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences), SEQ id no: the underlined portion of 71 encodes PDI SP-H3/Bri's F ' 1, E1, RB, E2, F ' 2, F, TMD/CT;

SEQ ID NO: 72 (construct 737; chimeric H5/H3 expression cassette comprising the CaMV35S promoter, CPMV-HT5 'UTR, chimeric hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences), SEQ ID NO: 72 underlined parts encode F '1, E1, RB, E2, F' 2, F, TMD/CT for PDI SP-H5/Indo;

SEQ ID NO: 74 (construct 739; HA expression cassette comprising the CaMV35S promoter, CPMV-HT5 'UTR, PDI signal peptide coding sequence, hemagglutinin coding sequence of HA type B/Florida/4/06, CPMV-HT 3' UTR, and NOS terminator sequences), SEQ ID NO: the underlined portion of 74 encodes F '1, E1, RB, E2, F' 2, F, TMD/CT of PDI SP-B/Flo;

SEQ ID NO: 75 (construct 734; chimeric H5/B expression cassette comprising CaMV35S promoter, CPMV-HT5 'UTR, chimeric hemagglutinin coding sequence, CPMV-HT 3' UTR, and NOS terminator sequences), SEQ ID NO: the 75 underlined parts encode TND/CT for F '1, E1, RB, E2, F' 2, F-H5/Indo for PDI SP-B/Flo.

The invention also includes methods of hybridizing to any one of SEQ ID NOs: 63-65, 68, 69 and 71-75 are underlined nucleotide sequences that hybridize. The invention also includes methods of hybridizing to any one of SEQ ID NOs: 63-65, 68, 69 and 71-75, the complementary sequences of the underlined portions. These are similar to SEQ ID NO: 63-65, 68, 69 and 71-75 underlined portions or SEQ ID NO: the nucleotide sequences to which the complementary sequences of the underlined portions 63-65, 68, 69 and 71-75 hybridize encode a chimeric hemagglutinin protein, the expression of which forms chimeric VLPs, and the chimeric VLPs induce antibody production when administered to a subject. For example, the nucleotide sequences are expressed in plant cells to form chimeric VLPs which can be used to produce antibodies capable of binding HA (including mature HA of one or more influenza types or subtypes, HA0, HA1, or HA2), which, when administered to a subject, induce an immune response.

Hybridization under stringent hybridization conditions is known in the art (see, e.g., Current protocols Molecular Biology, eds. Ausubel et al, 1995 and suppl., Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3 rd edition, 2001; each incorporated herein by reference). An example of such stringent hybridization conditions may be hybridization in 4 XSSC at 65 ℃ for about 16-20 hours followed by washing in 0.1 XSSC at 65 ℃ for 1 hour, or washing twice (20 or 30 minutes each) in 0.1 XSSC at 65 ℃. Alternatively, an exemplary stringent hybridization condition may be overnight (16-20 hours) in 50% formamide, 4 XSSC at 42 ℃ followed by 1 hour of washing in 0.1 XSSC at 65 ℃, or 2 times (20 or 30 minutes each) in 0.1 XSSC at 65 ℃, or overnight (16-20 hours), or in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO) at 65 ℃₄The pH of the buffer solution is 7.2; 10mM EDTA), 2 washes in 0.1 XSSC, 0.1% SDS at 50 deg.C (20 or 30 minutes each), or 2 washes in 2 XSSC, 0.1% SDS at 65 deg.C (20 or 30 minutes each).

Furthermore, the present invention includes nucleotide sequences characterized by a nucleotide sequence that is complementary to a nucleotide sequence encoding any one of SEQ id nos: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 71. SEQ ID NO: 72. SEQ ID NO: 74. SEQ ID NO: the nucleotide sequence of the 75 underlined portion of the chimeric HA HAs about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any amount of sequence identity or sequence similarity therebetween, wherein the nucleotide sequence encodes a hemagglutinin protein which, when expressed, forms a chimeric VLP which induces antibody production. For example, the nucleotide sequences are expressed in plant cells to form chimeric VLPs which can be used to produce antibodies capable of binding HA (including mature HA, HA0, HA1 or HA2), which when administered to a subject induce an immune response.

An "immune response" generally refers to the response of the acquired immune system. The adaptive immune system typically includes both humoral and cell-mediated responses. The humoral response is an immunological aspect mediated by the production of secreted antibodies by cells of the B lymphocyte lineage (B cells). Secreted antibodies bind to antigens on the surface of invading microorganisms (e.g., viruses or bacteria) and label them for destruction. Humoral immunity generally refers to the processes of and associated with antibody production, as well as effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell formation, opsonin promotion of endocytosis, pathogen clearance, and the like. The term "modulate" refers to an increase or decrease in a particular response or parameter as determined by any assay commonly known or used, some of which are exemplified herein.

A cell-mediated response is an immune response that does not involve antibodies, but rather involves activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. Cell-mediated immunity generally refers to the activation of some Th cells, the activation of Tc cells, and T cell-mediated responses. Cell-mediated immunity is particularly important in response to viral infection.

For example, the ELISPOT assay can be used to measure induction of antigen-specific CD8 positive T lymphocytes; stimulation of CD4 positive T lymphocytes can be measured using a proliferation assay. The titer of anti-influenza virus antibodies can be quantified using an ELISA assay; anti-isotype antibodies (e.g., anti-IgG, anti-IgA, anti-IgE, or anti-IgM) can also be used to measure the isotype of antigen-specific or cross-reactive antibodies. Methods and techniques for performing such assays are well known in the art.

Hemagglutination inhibition (HI or HAI) assays may also be used to demonstrate the efficacy of antibodies induced by a vaccine or vaccine composition comprising chimeric HA or chimeric VLPs that inhibit Red Blood Cell (RBC) agglutination by recombinant HA. The hemagglutination-inhibiting antibody titer of serum samples can be assessed by microtiter HAI (Aymard et al, 1973). Any red blood cells from several species may be used, e.g., horses, turkeys, chickens, etc. This assay gives indirect information about the assembly of HA trimers on the surface of VLPs, confirming the correct display of HA antigenic sites.

Cross-reactive HAI titration can also be used to demonstrate the efficacy of immune responses against other strains of virus associated with vaccine subtypes. For example, sera of subjects immunized with a vaccine composition comprising a chimeric hemagglutinin (comprising HDC of a first influenza type or subtype) may be used with a second strain of whole virus or viral particle in an HAI assay and the HAI titer determined.

Without wishing to be bound by theory, the ability of HA to bind RBCs from different animals is driven by the affinity of HA for α 2, 3 or α 2, 6 bonded sialic acids and the presence of these sialic acids on the surface of RBCs. Equine and avian influenza virus HA agglutinates erythrocytes from all several species (including turkey, chicken, duck, guinea pig, human, sheep, horse and cattle); while human HA binds red blood cells of turkeys, chickens, ducks, guinea pigs, humans and sheep (Ito T. et al, 1997, Virology, vol. 227, 493 499; and Medeiros R et al, 2001, Virology, vol. 289, 74-85).

The presence or level of cytokines can also be quantified. For example, T helper cell responses (Th1/Th2) are characterized by measuring IFN-. gamma.and IL-4 secreting cells using ELISA (e.g., BDbiosciences OptEIA kit). Peripheral Blood Mononuclear Cells (PBMC) or spleen cells obtained from a subject can be cultured and the supernatant analyzed. T lymphocytes can also be quantified by fluorescence-activated cell sorting (FACS) using marker-specific fluorescent labeling and methods known in the art.

Microneutralization assays may also be performed to characterize immune responses in subjects, see, e.g., the methods of Rowe et al, 1973. Virus neutralization titers can be obtained by several methods, including: 1) after crystal violet fixation/staining of the cells, lysis spots were counted (plaque assay); 2) observing cell lysis in the culture under a microscope; 3) the NP viral protein (associated with viral infection of host cells) was detected by ELISA and spectrophotometry.

Sequence identity or sequence similarity can be determined using a sequence comparison program, such as that provided by DNASIS (e.g., using, but not limited to, the following parameters: gap penalty of 5, number of top diagonals (# of top diagonals) of 5, fixed gap penalty of 10, k-ary 2, gap of 10, window size of 5). However, other methods of sequence alignment for comparison are well known in the art, such as the Smith & Waterman algorithm (1981, adv. Appl. Math.2: 482), the Needleman & Wunsch algorithm (J.mol. biol.48: 443, 1970), the Pearson & Lipman algorithm (1988, Proc. nat' l. Acad. Sci. USA 85: 2444), and computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA and BLAST (Altschul et al, 1990.J.mol Biol 215: 403-410)), or by manual alignment and visual inspection. Nucleic acid or amino acid sequences can be compared or aligned and the consensus sequence determined using any of several software packages known in the art, such as MULTALIN (Corpet f., 1988, nucleic Acids res., 16(22), 10881-; alternatively, the sequences may be aligned manually and the similarity and differences between the sequences determined.

A fragment or portion of a protein, fusion protein or polypeptide comprises a peptide or polypeptide comprising a portion of the amino acid composition of a particular protein or polypeptide, provided that upon expression the fragment forms a chimeric VLP. For example, the fragment may comprise an antigenic region, a stress response inducing region, or a region comprising a functional domain of a protein or polypeptide. The fragments may also comprise regions or domains common to proteins of the same general family, or the fragments may comprise sufficient amino acid sequences to specifically identify the full-length protein from which they are derived.

For example, a fragment or portion may comprise from about 60% to about 100% of the full length of the protein, or any amount therebetween, provided that upon expression the fragment may form a chimeric VLP. For example, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, or any amount therebetween, of the full length of the protein. Alternatively, depending on the chimeric HA, the fragment or portion may be from about 150 to about 500 amino acids or any amount therebetween, provided that upon expression the fragment may form a chimeric VLP. For example, depending on the chimeric HA, the fragment or portion may be from about 150 to about 500 amino acids or any amount therebetween, from about 200 to about 500 amino acids or any amount therebetween, from about 250 to about 500 amino acids or any amount therebetween, from about 300 to about 500 amino acids or any amount therebetween, from about 350 to about 500 amino acids or any amount therebetween, from about 400 to about 500 amino acids or any amount therebetween, from about 450 to about 500 amino acids or any amount therebetween, provided that upon expression the fragment may form a chimeric VLP. For example, about 5,10, 20, 30, 40, or 50 amino acids or any amount therebetween may be removed from the C-terminus, N-terminus, or both the N and C-termini of the chimeric HA protein, provided that upon expression the fragment may form a chimeric VLP.

The numbering of amino acids in any given sequence is relative to that particular sequence, however, one skilled in the art can readily determine the "identity" of a particular amino acid in a sequence based on structure and/or sequence. For example, if the 6N-terminal amino acids are removed, this will change the specific coding identity of the amino acids (e.g., relative to the full length of the protein), but will not change the relative positions of the amino acids in the structure.

The present invention describes, but is not limited to, the expression of nucleic acids encoding chimeric HA in plants, plant parts or plant cells, and the production of chimeric influenza VLPs in plants suitable for vaccine production. Examples of such nucleic acids include, but are not limited to, e.g., SEQ ID NOs: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 71. SEQ ID NO: 72. SEQ ID NO: 74. SEQ ID NO: 75.

the invention also provides for the expression of nucleic acids encoding chimeric HA (such as, but not limited to, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75) in plants, plant parts or plant cells, and the production of candidate influenza vaccines or reagents comprising recombinant influenza structural proteins that self-assemble into functional and immunogenic homotypic macromolecular protein structures, including subviral influenza particles and chimeric influenza VLPs, in transformed plant cells.

Accordingly, the present invention provides chimeric VLPs, and methods of producing chimeric VLPs in a plant expression system by expressing a single chimeric envelope protein.

Nucleic acids encoding influenza subtype chimeric HA (e.g., SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75) can be synthesized by reverse transcription and Polymerase Chain Reaction (PCR) using HA RNA. For example, RNA may be isolated from H1/NC, H1/Bri, H3/Bri, B/Flo or H5/Indo or from cells infected with these or other influenza virus types or subtypes. For reverse transcription and PCR, HA RNA-specific oligonucleotide primers can be used. Alternatively, the nucleic acid encoding the chimeric HA may be chemically synthesized using methods known to those skilled in the art.

The present invention also relates to genetic constructs comprising a nucleic acid encoding a chimeric HA operably linked to regulatory elements operable in plants, as described above. Examples of regulatory elements useful in plant cells and useful in the present invention include, but are not limited to, those of plastocyanin regulatory region (US 7,125,978; incorporated herein by reference) or ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO; U.S. Pat. No.4,962,028; incorporated herein by reference), chlorophyll a/b binding protein (CAB; Leutwiler et al; 1986; incorporated herein by reference), ST-LS1 (related to the oxygen-releasing complex of photosystem II, described in Stockhaus et al 1987, 1989; incorporated herein by reference).

The gene constructs of the invention also comprise constitutive promoters which direct the expression of genes operably linked to the promoters in various parts of the plant and which are expressed continuously throughout the development of the plant. A non-limiting example of a constitutive promoter is that associated with the CaMV35S transcript (e.g., Odell et al, 1985, Nature, 313: 810-.

Examples of sequences comprising plastocyanin regulatory regions are SEQ ID NO: 58 to the underlined sequence encoding the PDI signal peptide. The regulatory element or region may enhance translation of a nucleotide sequence operably linked thereto, wherein the nucleotide sequence may encode a protein or polypeptide. Another example of a regulatory region is a regulatory region derived from the untranslated region of Cowpea Mosaic Virus (CPMV), which can be used to preferentially translate a nucleotide sequence operably linked thereto. This CPMV regulatory region is used in the context of an over-translatable CMPV-HT system, see, e.g., Sainsbury et al, 2008, Plant Physiology 148: 1212, 1218; sainsbury et al, 2008 Plant Biotechnology journal 6: 82-92, both incorporated herein by reference).

Accordingly, one aspect of the invention provides a nucleic acid comprising a regulatory region operably linked to a sequence encoding a chimeric influenza HA. The regulatory region may be a plastocyanin regulatory element and the chimeric influenza HA may comprise subdomains from H5/Indo, H1/Bri, H3/Bri, H1/NC, B/Flo influenza types, subtypes or strains. A nucleic acid sequence comprising a plastocyanin regulatory element and a chimeric influenza HA is identified herein by SEQ ID NO: 63, and 64. A nucleic acid sequence comprising a 35S regulatory element and a chimeric influenza HA is identified herein by SEQ ID NO: 68. 69 and 71-75.

In another aspect, the invention provides a nucleic acid comprising a CPMV regulatory region and a chimeric influenza HA (comprising a subdomain from H5/Indo, H1/Bri, H3/Bri, H1/NC, B/Flo influenza type, subtype or strain). The nucleic acid sequence comprising CPMP regulatory elements and chimeric HA is described herein by SEQ ID NO: 66-69 and 71-75.

Chimeric influenza VLPs produced in plants bud from the plasma membrane, and thus the lipid composition of chimeric VLPs reflects the type of plant cell or plant tissue from which they are produced. The VLPs produced according to the invention comprise chimeric HA of two or more types or subtypes of influenza, complexed with plant-derived lipids. Plant lipids stimulate specific immune cells and enhance the induced immune response.

Plant lipids such as PC (phosphatidylcholine) and PE (phosphatidylethanolamine) and glycosphingolipids can bind to CD1 molecules expressed by mammalian immune cells (e.g., Antigen Presenting Cells (APCs), such as dendritic cells and macrophages) and other cells, including B-lymphocytes and T-lymphocytes in the thymus and liver. (for review see Tsuji M.2006 Cell Mol Life Sci 63: 1889-98). The CD1 molecule is similar in structure to the Major Histocompatibility Complex (MHC) class I molecule, and functions to present glycolipid antigens to NKT cells (natural killer T cells). Upon activation, NKT cells activate innate immune cells (e.g., NK cells and dendritic cells) and also activate adaptive immune cells (e.g., antibody-producing B cells and T cells).

Phytosterols present in influenza VLPs complexed with lipid bilayers (e.g., plasma membrane-derived envelopes) may provide advantageous vaccine compositions. Without wishing to be bound by theory, plant-produced VLPs (including VLPs comprising chimeric HA) complexed with lipid bilayers (e.g., plasma membrane-derived envelopes) may induce a stronger immune response than VLPs made in other expression systems and may be similar to the immune response induced by live or attenuated whole virus vaccines.

Thus, in some embodiments, the present invention provides a VLP (comprising a chimeric HA) complexed with a plant-derived lipid bilayer. In some embodiments, the plant-derived lipid bilayer may comprise the envelope of the VLP.

VLPs produced in plants may comprise chimeric HA containing plant-specific N-glycans. Accordingly, the present invention also provides VLPs comprising chimeric HA having plant-specific N-glycans.

Furthermore, modification of N-glycans in plants is known (see, e.g., WO 2008/151440; which is incorporated herein by reference) and chimeric HA containing modified N-glycans can be produced. HA can be obtained that comprise N-glycans with modified glycosylation patterns (e.g., reduced fucosylation, reduced xylosylation, or reduced both fucosylation and xylosylation), or chimeric HA can be obtained that contain modified glycosylation patterns, wherein the protein lacks fucosylation, xylosylation, or both, and comprises increased galactosylation. Furthermore, modulation of post-translational modifications (e.g., terminal addition of galactose) can result in reduced fucosylation and xylosylation of the expressed chimeric HA as compared to wild-type plants expressing the chimeric HA.

For example, and not to be considered as limiting, synthesis of a chimeric HA having a modified glycosylation pattern can be achieved by co-expressing a protein of interest with a nucleotide sequence encoding a β -1, 4-galactosyltransferase (GalT), such as but not limited to a mammalian GalT or a human GalT, although galts from other sources can also be used. The catalytic domain of GalT can also be fused to the CTS domain (i.e., cytoplasmic tail, transmembrane domain, stem region) of N-acetylglucosaminyltransferase (GNT1) to produce a GNT1-GalT hybrid enzyme, and the hybrid enzyme can be co-expressed with HA. HA may also be co-expressed with a nucleotide sequence encoding N-acetylglucosaminyltransferase III (GnT-III), such as but not limited to mammalian GnT-III or human GnT-III, although GnT-III from other sources may also be used. In addition, GNT1-GnT-III hybrid enzymes comprising a CTS of GNT1 fused to GnT-III may also be used.

Accordingly, the invention also includes a VLP comprising a chimeric HA having modified N-glycans.

Without wishing to be bound by theory, the presence of plant N-glycans on the chimeric HA may stimulate an immune response by promoting the binding of antigen presenting cells to the HA. Saint-jore-Dupas et al (TrendsBiotechnol 25: 317-23, 2007) have proposed the use of plant N-glycans to stimulate an immune response. Furthermore, the conformation of the VLPs may be advantageous for antigen presentation and enhance the adjuvant effect of the VLPs when complexed with a lipid layer of plant origin.

"regulatory region", "regulatory element" or "promoter" refers to a portion of a nucleic acid, which may be composed of DNA or RNA or both DNA and RNA, that is usually (but not always) located upstream of the protein coding region of a gene. When the regulatory region is active and operably linked or operably linked to the gene of interest, it results in the expression of the gene of interest. Regulatory elements may mediate organ-specific, or control the activation of developmental or temporal genes. "regulatory region" includes promoter elements, core promoter elements that exhibit the basic activity of a promoter, elements that can be induced in response to an external stimulus, elements that mediate promoter activity (e.g., negative regulatory elements or transcriptional enhancers). "regulatory region" as used herein also includes elements which are active after transcription, such as regulatory elements which regulate gene expression, for example translational enhancers and transcriptional enhancers, translational repressors and transcriptional repressors, upstream activating sequences and mRNA instability determinants (mRNA instability determinants). Several of these latter elements may be located adjacent to the coding region.

In the present disclosure, the term "regulatory element" or "regulatory region" refers generally to a DNA sequence, usually (but not always) located upstream (5') of a coding sequence of a structural gene, which controls the expression of the coding region by providing recognition by RNA polymerase and/or other factors required for transcription to initiate at a particular site. However, it is understood that other nucleotide sequences located within introns or 3' of the sequence may also contribute to the regulation of expression of the coding region of interest. One example of a regulatory element that provides recognition for RNA polymerase or other transcription factor to ensure initiation at a particular site is a promoter element. Most (but not all) eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence consisting of adenosine and thymidine nucleotide base pairs, typically located about 25 base pairs upstream of the transcription start site. Promoter elements include the basic promoter elements responsible for initiating transcription as well as other regulatory elements (as described above) that regulate gene expression.

There are several types of regulatory regions, including developmentally regulated, inducible or constitutive. The developmentally regulated regulatory regions or regulatory regions that control differential expression of the genes being controlled are activated in a particular organ or tissue of an organ at a particular time during development of the organ or tissue. However, some regulatory regions that are developmentally regulated may also preferentially be active at particular developmental stages in certain organs or tissues, they may also be active in a developmentally regulated manner, or at basal levels within other organs or tissues of the plant. Examples of tissue-specific regulatory regions (see, e.g., specific regulatory regions) include the napin promoter and the cruciferin promoter (Rask et al, 1998, J.plant Physiol.152: 595-. Examples of leaf-specific promoters include the plastocyanin promoter (see, e.g., SEQ ID NO: 58); US 7,125,978, which is incorporated herein by reference.

An inducible regulatory region is a regulatory region capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequence or gene will not be transcribed. In general, a protein factor that specifically binds to an inducible regulatory region to activate transcription can exist in an inactive form and then be converted, directly or indirectly, to an active form by an inducer. However, protein factors may also be absent. The inducer may be a chemical agent, such as a protein, metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress exerted directly by heat, cold, salt or toxic elements, or indirectly by the action of a pathogen or pathogenic agent (e.g. a virus). Plant cells containing inducible regulatory regions can be exposed to an inducer by applying the inducer to the cell or outside the plant (e.g., by spraying, watering, heating, or the like). Inducible regulatory elements can be derived from Plant genes or non-Plant genes (e.g., Gatz, C. and Lenk, I.R.P., 1998, Trends Plant Sci.3, 352-358, which is incorporated herein by reference). Examples of inducible promoters that can be used include, but are not limited to, tetracycline-inducible promoters (Gatz, C., 1997, Ann. Rev. Plant physiol. Plant mol. biol.48, 89-108, which are incorporated herein by reference), steroid-inducible promoters (Aoyama, T. and Chua, N.H., 1997, Plant J.2, 397-404, which are incorporated herein by reference) and ethanol-inducible promoters (Salter, M.G et al, 1998, Plant journal 16, 127-132; Caddick, M.X. et al, 1998, Nature Biotech.16, 177-180, which are incorporated herein by reference), cytokinin-inducible IB6 and CKI1 genes (Brandstatter, I. and Kieber, J.J., 1998, Plant Cell 10, 1009-.

Constitutive regulatory regions direct the expression of genes in various parts of the plant and continue throughout plant development. Examples of known constitutive regulatory elements include promoters associated with: CaMV35S transcripts (Odell et al, 1985, Nature, 313: 810-. The term "constitutive" as used herein does not necessarily mean that the gene under the control of the constitutive regulatory region is expressed at the same level in all cell types, but rather that the gene is expressed in multiple cell types, although different abundances are often observed. Constitutive regulatory elements may be coupled to other sequences to further enhance transcription and/or translation of the nucleotide sequence to which they are operably linked. For example, the CMPV-HT system is derived from the untranslated region of cowpea mosaic virus (CPMV) and is shown to enhance translation of the relevant coding sequence.

"native" refers to a nucleic acid or amino acid sequence that is naturally occurring, or "wild-type".

"operably linked" refers to a particular sequence (e.g., a regulatory element and a coding region of interest) that interacts, directly or indirectly, to achieve a predetermined function (e.g., mediate or regulate gene expression). For example, the interaction between operably linked sequences may be mediated by a protein that interacts with the operably linked sequences.

One or more nucleotide sequences of the invention may be expressed in any suitable plant host transformed with a nucleotide sequence, or construct or vector of the invention. Examples of suitable hosts include, but are not limited to, agricultural crops including alfalfa, canola, Brassica spp, corn, tobacco, nicotiana spp, alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, and the like.

One or more chimeric gene constructs of the invention may further comprise a 3' untranslated region. By 3' untranslated region is meant a portion of a gene that comprises a segment of DNA that contains a polyadenylation signal and any other regulatory signals capable of affecting mRNA processing or gene expression. Polyadenylation signals are generally characterized by the addition of a polyadenylation chain to the 3' terminus of the mRNA precursor. Polyadenylation signals are often identified by the presence of a homologue of the classical form 5 'AATAAA-3', but variants may also occur.

Non-limiting examples of suitable 3 'regions are the untranslated regions of the 3' transcript containing the polyadenylation signal of the following genes: agrobacterium tumorigenic (Ti) plasmid genes (e.g., nopaline synthase (Nos gene)) as well as plant genes (e.g., soybean storage protein gene), the small subunit gene of ribulose-1, 5-bisphosphate carboxylase (ssRUBISCO; U.S. Pat. No.4,962,028, which is incorporated herein by reference), promoters for regulating plastocyanin expression (described in U.S. Pat. No. 7,125,978, which is incorporated herein by reference).

One or more chimeric gene constructs of the invention may further comprise an enhancer, which may be a translational or transcriptional enhancer, as desired. Enhancers can be located 5 'or 3' to the transcribed sequence. Enhancer regions are well known to those of skill in the art and may include the ATG initiation codon, adjacent sequences, and the like. The start codon, if present, can be in frame (phase) of the coding sequence to provide for proper translation of the transcribed sequence.

To aid in the identification of transformed plant cells, the constructs of the invention may be further processed to include a plant selectable marker. Useful selectable markers include enzymes that provide resistance to chemicals (e.g., antibiotics such as gentamicin, hygromycin, kanamycin; or herbicides such as phosphinothricin, glyphosate, chlorsulfuron, etc.). Similarly, enzymes that produce compounds that can be identified by a color change (e.g., GUS (β -glucuronidase)) or enzymes that provide luminescence (e.g., luciferase or GFP) can be used.

Also considered as part of the invention are transgenic plants, plant cells or seeds comprising the chimeric gene constructs of the invention. Methods for regenerating whole plants from plant cells are also known in the art. Generally, transformed plant cells are cultured in a suitable medium that may include a selection agent (e.g., an antibiotic) wherein a selectable marker is used to aid in the identification of the transformed plant cells. After callus formation, shoots can be promoted by the application of appropriate plant hormones according to known methods and transferred to rooting media to regenerate plants. The plant can then be used to establish reproducible generations, either by seed or using plant asexual propagation techniques. Transgenic plants can also be formed without the use of tissue cultures.

Also considered as part of the invention are transgenic plants, trees, yeast, bacteria, fungi, insects and animal cells comprising a chimeric gene construct containing a nucleic acid encoding a recombinant chimeric HA or HA0 of the invention for producing VLPs.

The regulatory elements of the invention may also be combined with coding regions of interest for expression in a variety of host organisms that may be used for transformation or transient expression. Such organisms include, but are not limited to, plants (monocots and dicots), such as, but not limited to, maize, cereals, wheat, barley, oats, nicotiana, brassica, soybean, kidney bean, pea, alfalfa, potato, tomato, ginseng, and arabidopsis thaliana.

Methods for stable transformation and regeneration of these organisms have been established in the art and are known to those skilled in the art. The method of obtaining transformed plants and regenerating plants is not critical to the present invention.

"transformation" refers to the transfer of genetic information (nucleotide sequences) between species that is either genotypic, phenotypic, or both. The interspecies transfer of genetic information from the chimeric construct to the host may be heritable and the transfer of genetic information is considered stable, or the transfer may be transient and the transfer of genetic information is not heritable.

The term "plant matter" refers to any material derived from a plant. Plant matter may include whole plants, tissues, cells, or any portion thereof. In addition, the plant matter may include intracellular plant components, cell explant components, liquid or solid extracts of plants, or combinations thereof. Further, the plant matter can include a plant, plant cell, tissue, liquid extract, or a combination thereof from a plant leaf, stem, fruit, root, or a combination thereof. The plant matter may comprise a plant or part thereof that has not been subjected to any treatment step. A part of a plant may comprise plant matter. However, it is also contemplated that the plant material may be subjected to the minimum treatment steps or more stringent treatments defined below, including partial or bulk protein purification using techniques well known in the art, including but not limited to chromatography, electrophoresis, and the like.

The term "minimal treatment" refers to partial purification of a plant material (e.g., a plant or portion thereof) comprising a protein of interest to obtain a plant extract, a homogenate, a fraction of a plant homogenate, etc. (i.e., minimal treatment). Partial purification may include, but is not limited to, disrupting plant cell structure to produce a composition containing soluble plant components and insoluble plant components, which may be separated by, for example, but not limited to, centrifugation, filtration, or a combination thereof. In this regard, proteins secreted in the extracellular space of leaves or other tissues can be easily obtained using vacuum or centrifugal extraction, or can be squeezed or released from the extracellular space by performing tissue extraction under pressure by passing through rollers or grinding or the like. Minimal processing may also include the preparation of crude extracts of soluble proteins, as these preparations will have negligible contamination from secondary plant products. Alternatively, minimal treatment may include aqueous extraction of soluble protein from the leaves followed by precipitation with any suitable salt. Other methods may include large scale maceration and juice extraction to allow direct use of the extract.

Plant matter (in the form of plant material or tissue) can be delivered orally to a subject. The plant matter may be administered with other foods as part of a dietary supplement or encapsulated. The plant matter or tissue may also be concentrated to improve or enhance palatability, or provided with other materials, ingredients, or pharmaceutical excipients as desired.

Examples of subjects or target organisms to which the VLPs of the invention may be administered include, but are not limited to, humans, primates, birds, waterfowls, migratory birds, quails, ducks, geese, poultry, chickens, pigs, sheep, equines, horses, camels, canines, dogs, felines, cats, tigers, leopards, muskrats, minks, ferrets, pets, livestock, rabbits, mice, rats, guinea pigs or other rodents, seals, whales and the like. These target organisms are exemplary and are not to be considered as limiting the application and uses of the invention.

It is contemplated that plants containing or expressing VLPs containing chimeric HAs of some embodiments of the invention may be administered to a subject or target organism in a variety of ways, as desired and as appropriate. For example, chimeric HA derived from plants can be extracted in crude, partially purified, or purified form prior to use. If the chimeric HA is at least partially purified, it may be produced in edible plants or inedible plants. Furthermore, if the chimeric HA is administered orally, plant tissue may be collected and consumed directly to the subject, or the collected tissue may be dried prior to consumption, or the animal may be allowed to feed on the plant without prior collection. Also considered within the scope of the invention is the use of the collected plant tissue as a food supplement for animal feed. If the plant tissue fed to the animal is not or hardly subjected to further treatment, it is preferred that the applied plant tissue is edible.

Post-transcriptional gene silencing (PTGS) can be involved in limiting transgene expression in plants, and co-expression of a silencing suppressor (HcPro) from potyvirus can be used to combat specific degradation of transgenic mRNA (Brigneti et al, 1998). Alternative suppressor of silencing are well known in the art and may be used as described herein (Chiba et al, 2006, Virology 346: 7-14, which is incorporated herein by reference), such as, but not limited to, TEV-p1/HC-Pro (tobacco etch virus-p 1/HC-Pro), BYV-p21, p19 of tomato bushy stunt virus (TBSV p19), capsid protein of tomato shriveling virus (TCV-CP), 2b of cucumber mosaic virus (CMV-2b), p25 of potato X virus (PVX-p25), p11 of potato M virus (PVM-p11), p11 of potato S virus (PVS-p11), p16 of blueberry flaviviruses (BScV-p16), p23 of citrus tristeza virus (CTV-p23), p24 of grapevine leaf roll-related virus-2 (GLV-24), P10 of grape virus A (GVA-p10), p14 of grape virus B (GVB-p14), p10 of Angelica dahurica latent virus (Heracleum latex virus) (HLV-p10) or p16 of garlic common latent virus (GCLV-p 16). Thus, suppressor of silencing (such as, but not limited to, HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV p19, TCV-CP, CMV-2b, PVX-p25, PVM-p11, PVS-p11, BScV-p16, CTV-p23, GLRaV-2p24, GBV-p14, HLV-p10, GCLV-p16, or GVA-p10) can be co-expressed with a nucleic acid sequence encoding a protein of interest to further ensure high levels of protein production in plants.

Furthermore, VLPs produced as described herein do not comprise Neuraminidase (NA). However, if a VLP comprising HA and NA is desired, NA may be co-expressed with HA.

Thus, the invention also includes suitable vectors containing chimeric HA sequences suitable for use in stable or transient expression systems. Genetic information may also be provided in one or more than one construct. For example, a nucleotide sequence encoding a protein of interest can be introduced into one construct and a second nucleotide sequence encoding a protein that modifies glycosylation of the protein of interest can be introduced into a separate construct. These nucleotide sequences can then be co-expressed in plants. However, constructs comprising nucleotide sequences encoding both the protein of interest and a protein that modifies glycosylation of the protein of interest may also be used. In this case, the nucleotide sequence will comprise a first sequence comprising a first nucleic acid sequence encoding a protein of interest operably linked to a promoter or regulatory region and a second sequence comprising a second nucleic acid sequence encoding a protein that modifies the glycosylation pattern of the protein of interest operably linked to the promoter or regulatory region.

"Co-expression" refers to the expression of two or more nucleotide sequences in a plant and in the same tissue of the plant at about the same time. However, the nucleotide sequences need not be strictly expressed simultaneously. But rather the expression of two or more nucleotide sequences in such a way that the encoded products have an opportunity to interact. For example, a protein that modifies glycosylation of a protein of interest can be expressed before or during expression of the protein of interest such that glycosylation modification of the protein of interest occurs. Two or more nucleotide sequences can be co-expressed using a transient expression system, wherein the two or more sequences are introduced into a plant at about the same time under conditions suitable for expression of the two sequences. Alternatively, a platform plant (platform plant) containing one of the nucleotide sequences, e.g. a sequence encoding a protein modifying the glycosylation pattern of a protein of interest, may be transformed with additional sequences encoding the protein of interest in a transient or stable manner. In this case, the sequence encoding the protein that modifies the glycosylation pattern of the protein of interest may be expressed in the desired tissue at the desired developmental stage, or its expression may be induced using an inducible promoter, while the additional sequence encoding the protein of interest may be expressed in the same tissue under similar conditions to ensure co-expression of the nucleotide sequence.

The constructs of the invention may be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, infection, and the like. For a review of these techniques see, e.g., Weissbach and Weissbach, Methods for Plant Molecular Biology, academic Press, New York VIII, pp 421-; geierson and Corey, plant molecular Biology, 2 nd edition (1988) and Miki and Iyer, fundametals of GeneTransfer in plants plant Metabolism, 2 nd edition, DT. Dennis, DH Turpin, DDLefebry, DB Layzell (eds.), Addison Wesley, Langmans Ltd. London, p. 561-. Other methods include direct DNA uptake, use of liposomes, electroporation (e.g., using protoplasts), microinjection, microprojectiles or whisker, and vacuum infection. See, e.g., Bilang et al (Gene 100: 247-: 70-73(1987), Howell et al (Science 208: 1265, 1980), Horsch et al (Science 227: 1229-: 694-: 343-; U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 6,403,865, respectively; 5,625,136 (all of which are incorporated herein by reference).

The constructs of the invention can be expressed using transient expression Methods (see Liu and Lomonosoff, 2002, Journal of viral Methods, 105: 343-348, which is incorporated herein by reference). Alternatively, vacuum-based transient expression methods can be used, such as Kapila et al 1997 Plant Science 122: 101-108 (which is incorporated herein by reference). These methods may include, for example, but are not limited to, agrobacterium inoculation or agrobacterium infection, however, other transient methods as described above may also be used. When using Agrobacterium inoculation or Agrobacterium infection, a mixture of Agrobacterium containing the desired nucleic acid enters the intercellular space of a tissue, e.g., a leaf, an aerial part of a plant (including stem, leaf and flower), other parts of a plant (stem, root, flower) or the whole plant. After crossing the epidermis, the Agrobacterium infects the cells and transfers copies of the t-DNA into the cells. the t-DNA is transcribed in episomal form and the mRNA is translated such that the protein of interest is produced in the infected cell, however, the passage of the t-DNA in the nucleus is transient.

The VLPs comprising chimeric HA provided by the present invention may be used in combination with existing influenza vaccines to supplement the vaccine to make it more effective, or to reduce the required dose administered. As known to those skilled in the art, a vaccine may be directed against one or more influenza viruses. Examples of suitable vaccines include, but are not limited to, vaccines commercially available from Sanofi-Pasteur, ID Biomedical, meridian, Sinovac, Chiron, Roche, MedImmune, GlaxoSmithKline, Novartis, Sanofi-Ayentis, Serono, fire Pharmaceuticals, and the like.

The VLPs of the invention may be mixed with suitable adjuvants known to those skilled in the art, as desired. Furthermore, the VLPs may be used in vaccine compositions containing an effective dose of VLPs for treating the above-mentioned target organisms. Furthermore, the VLPs produced according to the present invention may be combined with VLPs obtained using different influenza proteins, such as Neuraminidase (NA).

Accordingly, the present invention provides a method for inducing immunity to influenza virus infection in an animal or target organism comprising administering an effective dose of a vaccine comprising one or more than one VLP. The vaccine may be administered orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

The compositions of various embodiments of the present invention may comprise VLPs of two or more influenza strains or subtypes. "two or more" means two, three, four, five, six, seven, eight, nine, ten or more strains or subtypes. The strains or subtypes indicated may be single subtypes (e.g., all H1N1, or all H5N1), or may be combinations of subtypes. Exemplary subtypes and strains include H5/Indo, H1/Bri, H1/NC, H3/Bri, B/Flo. The choice of strain and combination of subtypes may depend on the region to which the subject is likely to be exposed to influenza, the proximity of the animal species (e.g. waterfowl, agricultural animals (e.g. pigs), etc.) to the population to be immunized and the strain with which the animal species is carrying, exposed to, or likely to be exposed to, the prediction of antigenic drift within the subtype or strain, or a combination of these factors. Examples of combinations used in the past years can be found in databases maintained by the World Health Organization (WHO) (see URL: WHO. int/csr/diease/influenza/vacine recomansions 1/en).

Two or more VLPs may be expressed separately and subsequently the purified or semi-purified VLPs combined. Alternatively, the VLPs may be co-expressed in the same host (e.g., plant part, or plant cell). VLPs may be combined or produced in a desired ratio (e.g., approximately equal ratio), or may be combined such that one subtype or strain makes up the majority of VLPs in the composition.

Accordingly, the present invention provides a composition comprising VLPs of two or more strains or subtypes.

Also provided are products comprising the packaging material and a composition comprising VLPs comprising chimeric HA. The composition comprises a physiologically or pharmacologically acceptable excipient and the packaging material may comprise a label identifying the active ingredient (e.g. VLP) of the composition.

Also provided are kits comprising a composition comprising a nucleic acid encoding a chimeric HA described herein and instructions for using the nucleic acid to produce the chimeric HA or a VLP comprising the chimeric HA. The kit can be used to produce VLPs comprising chimeric HA, and the instructions can include, for example, information for expressing the nucleic acid in a plant or plant cell, instructions for harvesting and obtaining the VLP from a plant or plant tissue.

In another embodiment, a kit for the manufacture of a medicament is provided comprising a VLP comprising a chimeric HA and instructions for its use. The instructions may comprise a series of steps for preparing a medicament useful for inducing a therapeutic or prophylactic immune response in a subject to which it is administered. The kit may further comprise instructions for dosage concentrations, dosage intervals, preferred methods of administration, and the like.

The present invention will be illustrated in more detail by the following examples. It should be understood, however, that these examples are for illustrative purposes only and should not be used in any way to limit the scope of the present invention.

The sequences described herein are summarized below.

Materials and methods

1.Assembly of HA expression cassettes

A-pCAMBIAPlasto

All manipulations were done using the general molecular biology protocol of Sambrook and Russell (2001; which is incorporated herein by reference). Table 1 shows the oligonucleotide primers used for expression cassette assembly. The first cloning step is the assembly of an acceptor plasmid containing the upstream and downstream regulatory elements of the alfalfa plastocyanin gene. The plastocyanin promoter and 5' UTR sequences were amplified from alfalfa genomic DNA using oligonucleotide primers XmaI-pPlas.c (SEQ ID NO: 1) and SacI-ATG-pPlas.r (SEQ ID NO: 2). The resulting amplification product was digested with XmaI and SacI and ligated with pCAMBIA2300(Cambia, Canberra, Australia) previously digested with the same enzymes to form pCAMBIApromoPlassto. Similarly, the 3' UTR sequence of the plastocyanin gene and the terminator were amplified from alfalfa genomic DNA using the primers SacI-PlasTer. c (SEQ ID NO: 3) and EcoRI-PlasTer. r (SEQ ID NO: 4), and the resulting product was digested with SacI and EcoRI and then inserted into the same site of pCAMBIApromoPlasto to form pCAMBIAPlasto.

B-Plasto-Natural SP-H5A/Indonesia/5/05 (construct No. 660)

Epoch Biolabs (Sugar Land, TX, USA) synthesized fragments encoding hemagglutinin of influenza strain A/Indonesia/5/05 (H5N 1; accession number LANL ISDN 125873). The resulting fragment containing the entire H5 coding region was shown (SEQ ID NO: 52, FIG. 17) as containing the native signal peptide flanked by a HindIII site immediately upstream of the initiating ATG and a SacI site immediately downstream of the stop codon (TAA). The H5 coding region was cloned into a plastocyanin-based expression cassette by PCR-based ligation as shown by Darveau et al (1995). Briefly, a first PCR amplification was performed using the primers Plato-443c (SEQ ID NO: 5) and SpHA (Ind) -Plasto.r (SEQ ID NO: 6) with pCAMBIApromoPlasto as a template. In parallel, a second amplification was performed using the primers Plasto-SpHA (SEQ ID NO: 7) and HA (Ind) -Sac.r (SEQ ID NO: 8) with the H5 coding fragment (SEQ ID NO: 52; FIG. 17) as template. The amplicons from the two reactions were mixed, and a third reaction (assembly reaction) was carried out using Plato-443c (SEQ ID NO: 5) and HA (Ind) -Sac.r (SEQ ID NO: 8) as primers using the mixture as a template. The resulting fragment was digested with BamHI (in the plastocyanin promoter) and SacI (at the 3' end of the fragment) and cloned into pCAMBIAPlasto previously digested with the same enzymes. The resulting plasmid was designated 660 and is shown in FIG. 18 (SEQ ID NO: 53).

C-Plasto-PDI SP-H1A/New Clintonia/20/99 (construct No. 540)

The open reading frame of the influenza strain A/New Canidonia/20/99 (H1N1) H1 gene was synthesized as two fragments (Plant Biotechnology Institute, National Research Council, Saskatoon, Canada). The first fragment synthesized corresponds to the wild-type H1 coding sequence lacking the 5 'signal peptide coding sequence and the 3' transmembrane domain coding sequence (GenBank accession AY 289929; SEQ ID NO: 54; FIG. 19). The 5 'end of the fragment consists of the terminal nucleotide encoding PDISP (containing the BglII restriction site), and a SacI/StuI double site was added immediately downstream of the 3' stop codon of the fragment, giving the amino acid sequence of SEQ ID NO: 55 (fig. 20). A second fragment encoding the C-terminus of the H1 protein (comprising a transmembrane domain and a cytoplasmic tail) was also synthesized, from the KpnI site to the stop codon, flanked 3' by SacI and StuI restriction sites (SEQ ID NO. 56; FIG. 21).

The first H1 fragment was digested with BglII and SacI and cloned into the same site of a binary vector (pCAMBIAPlasto) containing the plastocyanin promoter and 5' UTR fused to the signal peptide of the alfalfa Protein Disulfide Isomerase (PDI) gene (nucleotides 32-103; accession number Z11499; SEQ ID NO: 57; FIG. 22) to give a PDI-H1 chimeric gene located downstream of the plastocyanin regulatory element. The sequence of the plastocyanin-based expression cassette is shown in SEQ ID NO.58 (FIG. 23), which contains the promoter and PDI signal peptide upstream of the BglII restriction site, and the plastocyanin terminator downstream of the SacI site. The C-terminus of the H1 coding region (encoding the transmembrane domain and cytoplasmic tail) was added by inserting a synthetic fragment (SEQ ID NO: 56; FIG. 21) previously digested with KpnI and SacI into the H1 expression plasmid. The resulting construct, designated 540, is shown in SEQ ID NO.59 (FIG. 24).

D-Plasto-Natural SP-H1A/British Band/59/07 (construct No. 774)

Expression cassette number 774, which directs the expression of H1 of a/brisban/59/07, was assembled according to the following procedure. A synthetic fragment was synthesized comprising the entire hemagglutinin coding sequence (from ATG to termination), flanked at the 3' end by the alfalfa plastocyanin gene sequence starting with a DraIII restriction site corresponding to the first 84 nucleotides upstream of the plastocyanin ATG. The synthetic fragment also contains a SacI site immediately downstream of the stop codon.

The synthetic fragment was synthesized by Top Gene Technologies (Montreal, QC, Cahada). The synthesized fragment is shown in SEQ ID NO.60 (FIG. 25). For the assembly of the complete expression cassette, the synthetic fragment was digested with DraIII and SacI and cloned into pCAMBIAPlasto previously digested with the same enzymes, giving construct 774(SEQ ID NO. 61; FIG. 26).

E-CPMV HT-LC C51 (construct No. 828)

The CPMV-HT expression cassette uses the 35S promoter to control expression of mRNA containing a coding sequence of interest that is 5 ' flanked by nucleotides 1-512 from cowpea mosaic virus (CPMV) RNA2 (which has mutated ATGs at positions 115 and 161) and 3 ' flanked by nucleotides 3330-3481 from CPMV RNA2 (corresponding to the 3 ' UTR) followed by the NOS terminator. Plasmid pBD-C5-1LC (Sainsbury et al, 2008; plant Biotechnology Journal 6: 82-92 and PCT publication WO 2007/135480) was used to assemble CPMV-HT-based hemagglutinin expression cassettes. Mutation of ATG at positions 115 and 161 of CPMV RNA2 was achieved using a PCR-based ligation method (Darveau et al, Methods in Neuroscience 26: 77-85 (1995)). Two separate PCRs were performed using pBD-C5-1LC as a template. The primers used for the first amplification were pBinPlus.2613c (SEQ ID NO: 9) and Mut-ATG115R (SEQ ID NO: 10). The primer used for the second amplification is Mut-ATG161C (SEQ ID NO: 11) and LC-C5-1.110r (SEQ ID NO: 12). The obtained two fragments were mixed and used as a template for the third amplification using primers pBinPlus.2613C (SEQ ID NO: 9) and LC-C5-1.110r (SEQ ID NO: 12). The resulting fragment was digested with PacI and ApaI and cloned into pBD-C5-1LC digested with the same enzymes. The resulting construct is designated 828, and is shown in FIG. 27 (SEQ ID NO: 62).

F-H5A/H1A/Brisbane/59/07 Receptor Binding (RB) domain in Indonesia/5/05 backbone (construct No. 690)

Chimeric HA was prepared using a PCR-based ligation method reported by Darveau et al (Methods in Neuroscience 26: 77-85(1995)) by replacing the RB domain in H5A/Indonesian/5/05 with the RB domain of H1A/Brisban/59/07. In the first round of PCR, the plastocyanin promoter segment was fused to the native signal peptide, and the F' 1 and E1 domains of H5A/Indonesia/5/05 were amplified using the primers Plasto-443c (SEQ ID NO: 5) and E1H1B-E1H5I.r (SEQ ID NO: 13) with the template construct number 660(SEQ ID NO: 53, FIG. 18). A second fragment containing the RB domain coding sequence of H1A/Brisban/59/07 was amplified using construct number 774(SEQ ID NO: 61; FIG. 26) as a template using primers E1H 5N-E1H1B.c (SEQ ID NO: 14) and E2H 5I-RB H1B.r (SEQ ID NO: 15). A third fragment containing the E2, F' 2, F, transmembrane and cytoplasmic domains of H5A/Indonesia/5/05 was amplified using primers RBH 1B-E2H 5I.c (SEQ ID NO: 16) and HA (Ind) -SacI.r (SEQ ID NO: 8) with construct number 660(SEQ ID NO 53; FIG. 18) as template. The amplification products were then mixed and used as templates for a second round of amplification (assembly reaction) using the primers Plasto 443c (SEQ ID NO: 5) and HA (Ind) -SacI.r (SEQ ID NO: 8). The resulting fragment was digested with BamHI (in the plastocyanin promoter) and SacI (after the stop codon) and cloned into construct No. 660(SEQ ID NO: 53; FIG. 18) previously digested with the same restriction enzymes, resulting in construct No. 690(SEQ ID NO: 63). This construct is shown in FIG. 28.

G-H5A/Indonesia/H1A/Brisban/59/07 esterase and receptor binding domain in 5/05 backbone (E1-RB-E2) (construct No. 691)

Chimeric HA was assembled by replacing the E1-RB-E2 domain in H5A/Indonesian/5/05 with the E1-RB-E2 domain of H1A/Brillisban/59/07 using a PCR-based ligation method reported by Darveau et al (Methods in Neuroscience 26: 77-85 (1995)). In the first round of PCR, the plastocyanin promoter segment was fused to the native signal peptide, and the F '1 domain of H5A/Indonesia/5/05 was amplified using the primers Plasto-443c (SEQ ID NO: 5) and E1H 1B-F' 1H5I.r (SEQ ID NO: 17) with the template construct number 660(SEQ ID NO: 53, FIG. 18). In parallel, the other two fragments were amplified. A second fragment containing the coding sequence of the E1-RB-E2 domain of H1A/Brisban/59/07 was amplified using the primers F '1H 5N-E1H1B.c (SEQ ID NO: 18) and F' 2H5I-E2H1B.r (SEQ ID NO: 19) with the template construct number 774(SEQ ID NO: 61; FIG. 26). For the third fragment, the F '2, F, transmembrane and cytoplasmic domains of H5A/Indonesia/5/05 were amplified using the primers E2H 1B-F' 2H5I.c (SEQ ID NO: 20) and HA (Ind) -SacI.r (SEQ ID NO: 8) with the template construct number 660(SEQ ID NO 53; FIG. 18). The amplification products were then mixed and used as templates for a second round of amplification (assembly reaction) using the primers Plasto 443c (SEQ ID NO: 5) and HA (Ind) -SacI.r (SEQ ID NO: 8). The resulting fragment was digested with BamHI (in the plastocyanin promoter) and SacI (after the stop codon) and cloned into construct No. 660(SEQ ID NO: 53; FIG. 18) previously digested with the same restriction enzymes, giving construct No. 691(SEQ ID NO: 64). This construct is shown in figure 29.

H-H1A/New Caledonia/H5A/Indonesia/5/05 Receptor Binding (RB) domain in 20/99 backbone (construct No. 696)

Chimeric HA was prepared by replacing the RB domain in H1A/Neocalli/20/99 with the RB domain of H5A/Indonesia/5/05 using a PCR-based ligation method reported by Darveau et al (Methods in Neuroscience 26: 77-85 (1995)). In a first round of PCR, the plastocyanin promoter segment was fused to the alfalfa protein disulfide isomerase signal peptide (PDISP; accession number Z11499, nucleotides 32-103 of SEQ ID NO: 57; FIG. 22), and the F' 1 and E1 domains of H1A/Neokaridonia/20/99 were amplified using primers Plasto-443c (SEQ ID NO: 5) and E1H5I-E1H1NC.r (SEQ ID NO: 21) using construct number 540(SEQ ID NO: 59, FIG. 24) as a template. A second fragment containing the RB domain coding sequence of H5A/Indonesia/5/05 was amplified using the construct number 660(SEQ ID NO: 53; FIG. 18) as template using primers E1H1NC-E1H5I.c (SEQ ID NO: 22) and E2H1NC-RB H5I.r (SEQ ID NO: 23). A third fragment containing the E2, F' 2, F, transmembrane and cytoplasmic domains of H1A/New Carlidonia/20/99 was amplified using the primers RB H5I-E2 H1NC. c (SEQ ID NO: 24) and HA-SacI. r (SEQ ID NO: 25) with construct number 540(SEQ ID NO: 59; FIG. 24) as template. The amplification products were then mixed and used as templates for a second round of amplification (assembly reaction) using the primers Plasto 443c (SEQ ID NO: 5) and HA-SacI.r (SEQ ID NO: 25). The resulting fragment was digested with BglII and SacI and cloned into construct No. 540(SEQ ID NO: 59, FIG. 24) previously digested with the same restriction enzymes to obtain construct No. 696(SEQ ID NO: 65). This construct is shown in FIG. 30.

I-Assembly of H1A/Brilliban/59/2007 into the CPMV-HT expression cassette (construct No. 732)

The HA coding sequence from H1A/Brillisban/59/2007 was cloned into CPMV-HT as follows. Restriction sites ApaI (immediately upstream of ATG) and StuI (immediately downstream of the stop codon) were added to the hemagglutinin coding sequence by PCR amplification using construct No. 774(SEQ ID NO: 61; FIG. 26) as template with primers ApaI-H1B.c (SEQ ID NO: 26) and StuI-H1B.r (SEQ ID NO: 27). The resulting fragment was digested with ApaI and StuI restriction enzymes and cloned into construct No. 828(SEQ ID NO: 62, FIG. 27) digested with the same enzymes. The resulting cassette is designated construct number 732(SEQ ID NO: 66, FIG. 31).

J-Assembly of SpPDI-H1A/Brilliban/59/2007 into a CPMV-HT expression cassette (construct No. 733)

The sequence encoding the alfalfa protein disulfide isomerase signal peptide (PDISP; SEQ ID NO: 57 nucleotides 32-103, FIG. 22; accession number Z11499) was fused to the HA0 coding sequence of H1 from A/Brillishift/59/2007 as described below, and the resulting fragment was cloned into CPMV-HT. The H1 coding sequence was amplified using primers SpPDI-H1B.c (SEQ ID NO: 28) and SacI-H1B.r (SEQ ID NO: 29) using construct 774(SEQ ID NO: 61, FIG. 26) as template. The resulting fragment was flanked 5 'and 3' by the last few nucleotides encoding PDISP (including the BglII restriction site) and a SacI restriction site, respectively, in the H1 coding sequence. This fragment was digested with BglII and SacI and cloned into construct No. 540(SEQ ID NO: 59, FIG. 24) previously digested with the same restriction enzymes. The cassette coding sequence designated construct No. 787(SEQ ID NO67) is shown in figure 32. Restriction sites ApaI (immediately upstream of ATG) and StuI (immediately downstream of the stop codon) were added to the hemagglutinin coding sequence by PCR amplification using construct number 787(SEQ ID NO: 67; FIG. 32) as template with primers ApaI-SpPDI.c (SEQ ID NO: 30) and StuI-H1B.r (SEQ ID NO: 27). The resulting fragment was digested with ApaI and StuI restriction enzymes and cloned into construct No. 828(SEQ ID NO: 62, FIG. 27) digested with the same enzymes. The resulting cassette was designated construct number 733(SEQ ID NO: 68, FIG. 33).

K-Assembly of H5A/H1A/British Band/59/07 Receptor Binding (RB) Domain in Indonesia/5/05 backbone in CPMV-HT expression cassette (construct No. 734)

The coding sequence of the chimeric HA (RB domain with H1A/brisbane/59/07 in the H5A/indonesia/5/05 backbone) was cloned into CPMV-HT as follows. Restriction sites ApaI (immediately upstream of ATG) and StuI (immediately downstream of the stop codon) were added to the chimeric hemagglutinin coding sequence by PCR amplification using construct 690(SEQ ID NO: 63; FIG. 28) as template with primers ApaI-H5(A-Indo).1c (SEQ ID NO: 31) and H5(A-Indo) -StuI.1707r (SEQ ID NO: 32). The resulting fragment was digested with ApaI and StuI restriction enzymes and cloned into construct No. 828(SEQ ID NO: 62, FIG. 27) digested with the same enzymes. The resulting cassette is designated construct number 734(SEQ ID NO: 69, FIG. 34).

L-Assembly of SpPDI-H3A/British Band/10/2007 into a CPMV-HT expression cassette (construct No. 736)

The sequence encoding the alfalfa PDI signal peptide was fused to HA0 from H3A/brisbane/10/2007 and cloned into CPMV-HT as described below. First, a synthetic fragment was synthesized comprising the entire hemagglutinin coding sequence (from ATG to termination), flanked 3' by the alfalfa plastocyanin gene sequence corresponding to the first 84 nucleotides upstream of the plastocyanin ATG (starting with a DraIII restriction site). The synthetic fragment also contains a SacI site immediately after the terminator. The synthetic fragment was synthesized by TopGene Technologies (Montreal, QC, Canada). The synthesized fragment is shown in seq id NO: 70 (fig. 35) and further used as a template for PCR-based ligation.

Next, the alfalfa Protein Disulfide Isomerase (PDISP) (nucleotides 32-103; accession number Z11499; SEQ ID NO: 57; FIG. 22) signal peptide was ligated together with the ApaI restriction site immediately upstream of the ATG and the StuI restriction site downstream of the stop codon to the HA0 coding sequence of H3 from A/Brisban/10/2007 as follows. PDISP was ligated to the H3 coding sequence using a PCR-based ligation method reported by Darveau et al (Methods in Neuroscience 26: 77-85 (1995)). In a first round of PCR, the PDISP signal peptide was amplified using the primers ApaI-SpPDI.c (SEQ ID NO: 30) and H3B-SpPDI.r (SEQ ID NO: 33) with construct 540(SEQ ID NO: 59; FIG. 24) as a template. In parallel, another fragment containing part of the coding sequence H3A/Brisban/10/2007 (from codon 17 to the stop codon) was amplified using the previously synthesized fragment (SEQ ID NO: 70; FIG. 35) as a template using primers SpPDI-H3B.c (SEQ ID NO: 34) and StuI-H3B.r (SEQ ID NO: 35). The amplification products were then pooled and used as templates for a second round of amplification (assembly reaction) using primers ApaI-SpPDI.c (SEQ ID NO: 30) and StuI-H3B.r (SEQ ID NO: 35). The resulting fragment was digested with ApaI and StuI restriction enzymes and cloned into construct No. 828(SEQ ID NO: 62, FIG. 27) digested with the same enzymes. The resulting cassette is designated construct number 736(SEQ ID NO: 71, FIG. 36).

M-Assembly of chimeric SpPDI-H3A/Britisban/10/2007 (extracellular domain) + H5A/Indonesian/5/2005 (TmD + Cyto tail) in CPMV-HT expression cassette (construct No. 737)

The sequence encoding the alfalfa PDI signal peptide was fused to the extracellular domain of H3A/brisbane/10/2007 and the transmembrane and cytoplasmic domains of H5A/indonesia/5/2005 as described below and cloned into CPMV-HT. The PDISP-H3 coding sequence was fused to the H5 transmembrane domain using a PCR-based ligation method reported by Darveau et al (Methods in Neuroscience 26: 77-85 (1995)). In the first round of PCR, a fragment comprising the PDISP signal peptide and the H3 Brisban extracellular domain was generated by amplification using primers ApaI-SpPDI.c (SEQ ID NO: 30) and TmD H5I-H3B.r (SEQ ID NO: 36) with construct number 736(SEQ ID NO: 71; FIG. 36) as template. In parallel, another fragment containing the transmembrane and cytoplasmic domains of H5 Indonesia was amplified using construct number 660(SEQ ID NO: 53; FIG. 18) as template using primers H3B-TmD H5I.c (SEQ ID NO: 37) and H5(A-Indo) -StuI.1707r (SEQ ID NO: 32). The amplification products were then mixed and used as templates for a second round of amplification (assembly reaction) using primers ApaI-SpPDI.c (SEQ ID NO: 30) and H5(A-Indo) -StuI.1707r (SEQ ID NO: 32). The resulting fragment was digested with ApaI and StuI restriction enzymes and cloned into construct No. 828(SEQ ID NO: 62, FIG. 27) digested with the same enzymes. The resulting cassette was designated construct number 737(SEQ ID NO: 72, FIG. 37).

N-Assembly of SpPDI-HA B/Florida/4/2006 into CPMV-HT expression cassette (construct No. 739)

The sequence encoding the alfalfa PDI signal peptide was fused to HA0 from HA B/brisbane/4/2006 as described below and cloned into CPMV-HT. First, a synthetic fragment was synthesized comprising the entire hemagglutinin coding sequence (from ATG to termination), flanked 3' by the alfalfa plastocyanin gene sequence corresponding to the first 84 nucleotides upstream of the plastocyanin ATG (starting with a DraIII restriction site). The synthetic fragment also contained a SacI restriction site immediately after the stop codon. This synthetic fragment was synthesized by Epoch Biolabs (Sugar Land, Texas, USA). The synthesized fragment is shown in SEQ ID NO73 (fig. 38) and further used as template for PCR-based ligation.

Next, the alfalfa Protein Disulfide Isomerase (PDISP) (SEQ ID NO: nucleotides 32-103 of 57; FIG. 22; accession number Z11499) signal peptide was ligated together with the ApaI restriction site immediately upstream of the ATG and the StuI restriction site downstream of the stop codon to the HA0 coding sequence of HA from B/Florida/4/2006 as follows. PDISP was ligated to the HA coding sequence using a PCR-based ligation method reported by Darveau et al (methods in Neuroscience 26: 77-85 (1995)). In the first round of PCR, the PDISP signal peptide was amplified using primers ApaI-SpPDI.c (SEQ ID NO: 30) and HBF-SpPDI.r (SEQ ID NO: 38) with construct number 540(SEQ ID NO: 59; FIG. 24) as a template. In parallel, another fragment containing part of the coding sequence (from codon 16 to the stop codon) of HA from B/Florida/4/2006 was amplified using the previously synthesized fragment (SEQ ID NO: 73; FIG. 38) as a template using primers SpPDI-H3B.c (SEQ ID NO: 39) and StuI-HBF.r (SEQ ID NO: 40). The amplification products were then mixed and used as templates for a second round of amplification (assembly reaction) using primers ApaI-SpPDI.c (SEQ ID NO: 30) and StuI-HBF.r (SEQ ID NO: 40). The resulting fragment was digested with ApaI and StuI restriction enzymes and cloned into construct No. 828(SEQ ID NO: 62, FIG. 27) digested with the same enzymes. The resulting cassette was designated construct number 739(SEQ ID NO: 74, FIG. 39). O-Assembly of chimeric SpPDI-HA B/Florida/4/2006 (extracellular domain) + H5A/Indonesian/5/2005 (TmD + Cyto tail) in the CPMV-HT expression cassette (construct No. 745).

The sequence encoding the alfalfa PDI signal peptide was fused to the HA B/Florida/4/2006 extracellular domain and the transmembrane and cytoplasmic domains of H5A/Indonesia/5/2005 as described below and cloned into CPMV-HT. The PDISP-B/Florida/4/2006 ectodomain coding sequence was fused to the H5 transmembrane and cytoplasmic domains using a PCR-based ligation method reported by Darveau et al (Methods in Neuroscience 26: 77-85 (1995)). In the first round of PCR, using primers ApaI-SpPDI.c (SEQ ID NO: 30) and TmD H5I-B Flo.r (SEQ ID NO: 41), a fragment comprising the PDISP signal peptide fused to the HA B/Florida/4/2006 extracellular domain was generated using construct number 739(SEQ ID NO: 74; FIG. 39) as template for amplification. In parallel, another fragment containing the H5 Indonesia transmembrane and cytoplasmic domains was amplified using construct number 660(SEQ ID NO: 53; FIG. 18) as template using primers B Flo-TmD H5I.c (SEQ ID NO: 42) and H5(A-Indo) -StuI.1707r (SEQ ID NO: 32). The amplification products were then mixed and used as templates for a second round of amplification (assembly reaction) using primers ApaI-SpPDI.c (SEQ ID NO: 30) and H5(A-Indo) -StuI.1707r (SEQ ID NO: 32). The resulting fragment was digested with ApaI and StuI restriction enzymes and cloned into construct No. 828(SEQ ID NO: 62, FIG. 27) digested with the same enzymes. The resulting cassette was designated construct number 745(SEQ ID NO: 75, FIG. 40).

P-Assembly of chimeric SpPDI-HA B/Florida/4/2006 + H5A/Indonesia/5/2005 (TmD + Cyto tail) in 2X35S-CPMV-HT expression cassette (construct No. 747)

The sequence encoding alfalfa PDI signal peptide was fused to the transmembrane and cytoplasmic domains of HA0 and H5A/Indonesia/5/2005 from HA B/Florida/4/2006 as described below and cloned into 2X 35S-CPMV-HT. Promoter switching was performed using the PCR-based ligation method reported by Darveau et al (Methods in Neuroscience 26: 77-85 (1995)). Use of the primer PacI-MCS-2X35S.c (SEQ ID NO: 89) and CPMV 5' UTR-2X35S.r(SEQ IDNO：90)：

The first fragment containing the 2X35S promoter (SEQ ID NO: 88: FIG. 50A) was amplified by PCR using a plasmid containing the 2X35S promoter as a template. In parallel, primers are used2X35SCPMV 5' UTR.c (SEO ID NO: 91) andApaI-M prot.r(SEO ID NO：92)：

a second PCR was performed using construct 745(SEQ ID NO: 75; FIG. 40) as a template. The two fragments obtained were then mixed and used as the primer PacI-MCS-2 X35S.c. (SEQ ID NO: 89) andApaItemplate for the second round of PCR (assembly reaction) of M prot. r (SEQ ID NO: 92). The resulting fragment was digested with PacI and ApaI and cloned into construct 745(SEQ ID NO: 75, FIG. 40) digested with the same enzymes. The expression cassette is designated construct 747(SEQ ID NO: 93), the sequence of which is shown in FIG. 50B.

2. Assembly of chaperonin expression cassettes

Two heat shock protein (Hsp) expression cassettes were assembled. In the first cassette, expression of Arabidopsis thaliana (Columbia ecotype) cytoplasmic HSP70 (Athsp 70-1 in Lin et al (2001) Cell stress and Chaperones 6: 201-. A second cassette was also assembled which contained the coding region of alfalfa cytoplasmic HSP40(MsJ 1; Frugis et al (1999) Plant Molecular Biology 40: 397-408) under the control of the chimeric Nir/Plant promoter.

First, an acceptor plasmid (acceptor plasmid) containing an alfalfa nitrite reductase promoter (Nir), a GUS reporter gene, and an NOS terminator in a plant binary vector was assembled. Plasmid pNir3K51 (previously described in U.S. Pat. No.6,420,548) was digested with HindIII and EcoRI. The resulting fragment was cloned into pCAMBIA2300(Cambia, Canberra, Australia) digested with the same restriction enzymes to give pCAMBIA-Nir3K 51.

The coding sequences for Hsp70 and Hsp40 were cloned into the recipient plasmid pCAMBIANir3K51, respectively, by a PCR-based ligation method (Darveau et al, Methods in Neuroscience 26: 77-85 (1995)).

For Hsp40, the Msj1 coding sequence (SEQ ID NO: 76; FIG. 41) was amplified by RT-PCR from alfalfa (Rangcrander ecotype) leaf total RNA using primers Hsp40Luz.1c (SEQ ID NO: 43) and Hsp40Luz-SacI.1272r (SEQ ID NO: 44). A second amplification was performed with primers Plasto 443c (SEQ ID NO: 5) and Hsp40Luz-Plasto. r (SEQ ID NO: 45) using construct 660(SEQ ID NO: 53; FIG. 18) as template. The PCR products were then mixed and used as a template for a third amplification (assembly reaction) using the primers Plasto-443c (SEQ ID NO: 5) and Hsp40Luz-SacI.1272r (SEQ ID NO: 44). The resulting fragment was digested with HpaI (in the plastocyanin promoter) and cloned into pCAMBIANir3K51 previously digested with HpaI (in the Nir promoter) and SacI and treated with T4DNA polymerase to generate blunt ends. The resulting clones were screened for correct orientation and sequenced to check sequence integrity. The resulting plasmid, designated R850, is shown in FIG. 42 (SEQ ID NO: 77). The coding region of Athsp70-1 was amplified from Arabidopsis thaliana leaf RNA by RT-PCR using primers Hsp70Ara.1c (SEQ ID NO: 46) and Hsp70Ara-SacI.1956r (SEQ ID NO: 47). A second amplification was performed with primers Plato-443c (SEQ ID NO: 5) and Hsp70Ara-Plasto.r (SEQ ID NO: 48) using construct 660(SEQ ID NO: 53, FIG. 18) as template. The PCR products were then mixed and used as a template for a third amplification (assembly reaction) using the primers Plasto 443c (SEQ ID NO: 5) and Hsp70ARA-SacI.1956r (SEQ ID NO: 47). The resulting fragment was digested with HpaI (in the plastocyanin promoter) and cloned into pCAMBIANir3K51 digested with HpaI (in the Nir promoter) and SacI and treated with T4DNA polymerase to generate blunt ends. The resulting clones were screened for correct orientation and sequenced to check sequence integrity. The resulting plasmid, designated R860, is shown in FIG. 43 (SEQ ID NO: 78).

The dual Hsp expression plasmids were assembled as follows. R860(SEQ ID NO: 78; FIG. 43) was digested with BsrBI (downstream of the NOS terminator), treated with T4DNA polymerase to generate blunt ends, and digested with SbfI (upstream of the chimeric NIR/Plasto promoter). The resulting fragment (chimeric Nir/Plasto promoter-HSP 70 coding sequence-Nos terminator) was cloned into R850(SEQ ID NO: 77; FIG. 42) previously digested with SbfI and SmaI, both located in the multiple cloning site upstream of the chimeric Nir/Plasto promoter. The resulting plasmid was designated R870, which is shown in FIG. 44 (SEQ ID NO: 79).

3. Assembly of other expression cassettes

HcPro expression cassette

The HcPro construct (35HcPro) was prepared as described in Hamilton et al (2002). All clones were sequenced to confirm the integrity of the constructs. Agrobacterium tumefaciens (Agrobacterium tumefaciens) (AGL 1; ATCC, Manassas, VA 20108, USA) was transformed with the plasmid by electroporation (Mattanovich et al, 1989). The integrity of all rhizobia agrobacterium strains was confirmed by restriction mapping.

P19 expression cassette

The coding sequence for the Tomato Bushy Stunt Virus (TBSV) p19 protein was ligated to the alfalfa plastocyanin expression cassette by a PCR-based ligation method (Darveau et al, Methods in Neuroscience 26: 77-85 (1995)). In a first round of PCR, a segment of the plastocyanin promoter was amplified with the primers Plasto 443c (SEQ ID NO: 5) and supP19-Plasto. r (SEQ ID NO: 49) using construct 660(SEQ ID NO: 53) as template. In parallel, construct 35S: another fragment containing The coding sequence of p19 was amplified using The primers supP19-1c (SEQ ID NO: 50) and supP19-SacI.r (SEQ ID NO: 51) with p19 (as described by Voinnet et al, The Plant Journal 33: 949-956 (2003)) as template. The amplification products were then mixed and used as templates for a second round of amplification (assembly reaction) using the primers Plasto 443c (SEQ ID NO: 5) and SupP19-SacI.r (SEQ ID NO: 51). The resulting fragment was digested with BamHI (in the plastocyanin promoter) and SacI (at the end of the p19 coding sequence) and cloned into construct No. 660(SEQ ID NO: 53; FIG. 18) previously digested with the same restriction enzymes, giving construct No. R472. Plasmid R472 is shown in figure 45.

Construct No. 443

Construct number 443 corresponds to pCAMBIA2300 (empty vector).

TABLE 1 oligonucleotide primers used for expression cassette assembly.

Table 2: agrobacterium strains for expressing influenza hemagglutinin with native or PDI signal peptide

Agrobacterium strains	Expressed HA	Signal peptide	Expression cassette
				AGL1/540	H1 (A/New Kalidonia/20/99)	PDI	Plastocyanin
AGL1/774	H1 (A/British class/59/2007)	Natural substance (such as natural gas)	Plastocyanin
				AGL1/787	H1 (A/British class/59/2007)	PDI	Plastocyanin
AGL1/732	H1 (A/British class/59/2007)	Natural substance (such as natural gas)	35S/CPMV-HT
				AGL1/736	H3 (A/British class/10/2007)	PDI	35S/CPMV-HT
AGL1/660	H5(A Indonesia/5/2005)	Natural substance (such as natural gas)	Plastocyanin
				AGL1/739	B (B/Florida/4/2006)	PDI	35S/CPMV-HT
AGLI/828	CPMV HT-LC C51	C51LC	35S/CPMV-HT
				AGLI/690	H1/Bris RB+H5/Indo SDC	Natural substance (such as natural gas)	Plasto
AGLI/691	H1/Bri E1-RB-E2+H5SDC	Natural substance (such as natural gas)	Plasto
				AGLI/696	H5/Indo RB+H1/NC SDC	PDI	Plasto
AGLI/733	H1/Bri	PDI	35S/CPMV-HT
				AGLI/734	H1/Bri RB+H5/Indo SDC	Natural substance (such as natural gas)	35S/CPMV-HT

AGLI/737	H3/Bri extracellular domain + H5/Indo TDC	PDI	35S/CPMV-HT
				AGLI/745	B/Flo extracellular domain _ H5/Indo TDC	PDI	35S/CPMV-HT
AGL1/747	B/Flo extracellular domain _ H5/Indo TDC	PDI	2X35S/CPMV-HT

4.Preparation, inoculation, Agrobacterium infection and harvesting of plant biomass

Nicotiana benthamiana (Nicotiana benthamiana) was grown from seeds on flat ground filled with commercial peat substrate. Plants were grown in the greenhouse with an 16/8 light cycle at a temperature schedule of 25 deg.C day/night 20 deg.C. After 3 weeks of sowing, individual seedlings were picked, transplanted into pots and grown in the greenhouse for a further 3 weeks under the same environmental conditions. Before transformation, terminal and axillary buds were removed by pinching off the buds of the plants or by chemically treating the plants at different times as described below.

In the presence of 10mM 2- [ N-morpholine]Agrobacterium transfected with each construct was cultured in YEB medium (pH 5.6) of ethanesulfonic acid (MES), 20. mu.M acetosyringone, 50. mu.g/ml kanamycin and 25. mu.g/ml carbenicillin, until OD₆₀₀Is 0.6 to 1.6. Agrobacterium suspensions were centrifuged and resuspended in infection medium (10mM MgCl.) before use₂And 10mM MES, pH 5.6). Syringe infection was performed as described by Liu and Lomonosoff (2002, Journal of viral Methods, 105: 343-. For vacuum infection, the Agrobacterium tumefaciens suspension was centrifuged, resuspended in infection medium, and stored at 4 ℃ overnight. On the day of infection, batch cultures were diluted in 2.5 culture volumes and warmed before use. Whole plants of nicotiana benthamiana or nicotiana tabacum (n.tabacum) were inverted in bacterial suspension in air-tight stainless steel jars for 2 minutes under vacuum of 20-40 torr. After syringe or vacuum infestation, the plants are moved back to the greenhouse for 4-5 days until harvest. Unless otherwise indicated, all infections were performed by 1: 1 co-infection with AGL1/35S-HcPro, except for the strain with the CPMV-HT cassette (which was 1: 1 co-infected with strain AGL 1/R472).

5. Leaf sampling and Total protein extraction

After cultivation, the aerial parts of the plants were harvested, frozen at-80 ℃ and broken into pieces. Total soluble proteins were extracted by homogenizing each sample of frozen crushed plant material in 3 volumes of cold 50mM Tris (pH 8.0)0.15M NaCl, 0.04% sodium metabisulfite and 1mM phenylmethylsulfonyl fluoride (Polytron). After homogenization, the slurry was centrifuged at 20,000g for 20 minutes at 4 ℃ and the clarified crude extract (supernatant) was used for analysis. The total protein content of the clarified crude extract was determined by Bradford assay (Bio-Rad, Hercules, Calif.) using bovine serum albumin as a reference standard.

6. Protein analysis and immunoblotting

Protein concentrations were determined by BCA protein assay (Pierce Biochemicals, Rockport IL). Proteins were separated by SDS-PAGE under reducing conditions and stained with Coomassie blue. The stained gel was scanned and density analysis was performed using ImageJ Software (NIH).

Proteins from SEC elution fractions were precipitated with acetone (Bollag et al, 1996), resuspended IN 1/5 volumes of equilibration/elution buffer, separated by SDS-PAGE under reducing conditions and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics Corporation, Indianapolis, IN) for immunodetection. Prior to immunoblotting, membranes were blocked with 5% skim milk in Tris buffered saline (TBS-T) and 0.1% Tween-20 at 4 ℃ for 16-18 hours.

Immunoblotting was performed by incubation with 2. mu.g/ml of the appropriate antibody (Table 6) (in 2% skim milk, 0.1% TBS-Tween 20). The secondary antibodies used for the chemiluminescent detection are shown in table 4, diluted in 2% skim milk, 0.1% TBS-Tween20 as indicated. Immunoreactive complexes were detected by chemiluminescence using luminol (Roche Diagnostics corporation) as a substrate. Using EZ-Link PlusActivation of horseradish peroxidase conjugation kit (Pierce, Rockford, IL) for horseradish peroxidase conjugation of human IgG antibodies. Inactivated whole virus (WIV) used as a Control for testing subtypes H1, H3, and B was purchased from National Institutes for Biological Standards and Controls (NIBSC).

Table 3: electrophoresis conditions, antibodies and dilutions for immunoblotting of expressed proteins

FII：Fitzgerald Industries International，Concord，MA，USA；

NIBSC：National Institute for Biological Standards and Control；

JIR：Jackson ImmunoResearch，West Grove，PA，USA；

ITC：Immune Technology Corporation，Woodside，N Y，USA；

7.Clarification and concentration before SEC

To improve resolution and increase the signal of the eluted fractions, the crude protein extract of the extract to be loaded onto size exclusion chromatography was clarified and concentrated using the following method. The extract was centrifuged at 70000g for 20 min at 4 ℃, the pellet washed twice by resuspension with 1 volume (compared to the initial extract volume) of extraction buffer (50mM Tris, pH8.0, 0.15M NaCl) and centrifuged at 70000g for 20 min at 4 ℃. The obtained pellet was resuspended in 1/3 volumes (compared to the original extract volume), and proteins (including VLPs) were pelleted by adding 20% (w/v) PEG3350 followed by incubation on ice for 1 hour. The precipitated proteins were recovered by centrifugation at 10000g for 20 min at 4 ℃ and resuspended in 1/15 volumes of extraction buffer (compared to the original extract volume). After complete resuspension of the protein, the insoluble material was precipitated by centrifugation at 20000g for 5 minutes at 4 ℃ and the clear supernatant was recovered.

8. Size exclusion color of protein extractsSpectrum

Pack 32ml Sephacryl^TMSize Exclusion Chromatography (SEC) columns of S-500 high resolution beads (S-500 HR: GE Healthcare, Uppsala, Sweden, cat # 17-0613-10) equilibrated with equilibration/elution buffer (50mM Tris (pH8), 150mM NaCl). 1.5mL of crude protein extract was loaded onto the column, and then the elution step was performed with 45mL of equilibration/elution buffer. The eluate was collected in 1.5mL fractions, the relative protein content of which was thus monitored by mixing 10. mu.L fractions with 200. mu.L of diluted Bio-Rad protein staining reagent (Bio-Rad, Hercales, Calif.). The column was washed with 2 column volumes of 0.2N NaOH, then 10 column volumes of 50mM Tris (pH8), 150mM NaCl and 20% ethanol. After each separation the column was calibrated with blue dextran 2000(GE HealthCare Bio-Science corp., Piscataway, NJ, USA). The elution profiles of blue dextran 2000 and host soluble protein were compared between each separation to ensure consistency of the elution profiles between the columns used.

Example 1: replacement strategy for RB and/or esterase domains on the stem of influenza subtypes

The RB subdomain of H5/Indo may be replaced with the RB subdomain of H1, H3 or B HA. The resulting chimeric HA provided SDC H5/Indo to form VLPs and displayed a RB subdomain comprising H1, H3 or B immunogenic sites. The H5/Indo RB subdomain may be inserted into the H1 stem (H1/NC). FIGS. 15A and 15B show the amino acid sequences of the subdomain fusion sites shown, with the amino acid sequences of the corresponding subdomains shown in FIGS. 2 (constructs 690, 734, 696 and 691) and tables 4 (constructs 900 and 745) and 5 (constructs 910, 920 and 930). The amino acid sequences shown in figure 2 and tables 4 and 5 do not comprise a signal peptide sequence.

Table 4 subdomains and chimeric influenza HA. A chimeric influenza HA comprising a heterologous RB subdomain.

Sequence SEQ ID NO: amino acids 1-92 of 105 are the F' 1+ E1 domain of H5/Indo; amino acids 93-259 are the RB head domain of H3/Brisbanb, and amino acids 260-548 are the E2+ F' 2 domain of H5/Indo.

Sequence SEQ ID NO: amino acids 1-92 of 106 are the F' 1+ E1 domain of H5/Indo; amino acids 93-276 are the RB head domain of B/Florida and amino acids 277-565 are the E2+ F' 2 domain of H5/Indo.

Table 5 subdomains and chimeric influenza HA. A chimeric influenza HA comprising a heterologous RB subdomain.

SEQ ID NO: amino acids 1-42 of 107 are the N-terminal F' 1 domain of H5/Indo; amino acids 43-228 are the E1-RB-E2 head domain of H3/Brisbanb, and amino acids 229-507 are the F' 2 domain of H5/Indo.

SEQ ID NO: amino acids 1-42 of 108 are the N-terminal F' 1 domain of H5/Indo; amino acids 43-281 are the E1-RB-E2 head domain of B/Florida; amino acids 282-556 are the F' 2 domain of H5/Indo.

SEQ ID NO: amino acids 1-42 of 109 are the N-terminal F' 1 domain of H1/NC; amino acids 43-273 are the E1-RB-E2 head domain of H5/Indo, and amino acids 274-548 are the F' 2 domain of H1/NC.

The fusion sites of each chimera are selected adjacent to (but not necessarily directly at) the N and C termini of each subdomain-without wishing to be bound by theory, these fusion sites are selected to maximize the stability of the chimeric HA. For example, structural and sequence conservation is observed at the N-terminus of the RB subdomain (Ha et al 2002, EMBO J.21: 865-875; incorporated herein by reference). The region of less variation in the primary sequence was found to be located in the C-F Y-P triplet 15 amino acids before the E1 subdomain. This cysteine is involved in the number 3 disulfide bridge conserved in HA (see fig. 46 and 47). This attachment at the cysteine may provide more suitable or superior stability to the chimeric HA relative to the native sequence. The C-terminus of RB provides conserved features: for example, in the alignment, a conserved serine residue at position 1 and an E2 subdomain that initiates with a beta sheet are observed in all HA (Ha et al, 2002, EMBO J.21: 865-875; which is incorporated herein by reference). Thus, the C-terminus of this RB can be fused to the starting amino acid of the beta sheet of the E2 subdomain. Also, for chimeras containing the RB subdomain of H1/NC, H1Bri, H3/Bri or B/Flo on H5/Indo SDC and chimeras (6 in total) containing the H5/Indo RB subdomain on H1SDC, there was no or no substantial change in the pattern of disulfide bridges, but a disulfide bridge (disulfide No. 8) was added to the hybrid HA of B RB on the H5 stem. Addition of this disulfide bridge should not interfere with the folding of HA (because it is located in the RB domain and cysteines are adjacent in the sequence) and can provide a more stable hybrid HA.

The E1-RB-E2 subdomain of the first influenza type is replaced with the E1-RB-E2 subdomain of the second influenza type. This arrangement allows the display of more amino acids of the second influenza type on the surface of H5-VLP. In this example, HDC of H1, H3, or B was placed on H5/InDosDC, and HDC of H5/Indo was placed on H1/NC SDC (Table 5).

The linkage of HDC was determined by conserved cysteine residues (constituting disulfide bridge No.6 in HA type a and disulfide bridge No. 7 in HA type B). The HDC linkage C-terminal to the E2 subdomain is determined by another conserved cysteine residue which constitutes a disulfide bridge No.6 (second amino acid of the F' 2 subdomain) of influenza type H1 or H3 on H5/Indo SDC or influenza type H5 on H1 SDC. For influenza b chimeras, a linkage is established at the first cysteine that constitutes a disulfide bridge No.4 (positions 4 amino acids apart on the F' 2 subdomain and conserved between HA). The disulfide bridge pattern of the resulting chimera did not show any changes — H1/H3/H5 hybrid HA contains 6 disulfide bridges and B hybrid HA HAs 7 disulfide bridges.

Example 2: replacement of the corresponding part of H5A/indonesia/5/05 with the Receptor Binding (RB) or receptor binding and esterase (E1-RB-E2) subdomain of H1A/brisban/59/2007: comparison of expression of chimeras and native forms.

To combine the high accumulation levels of VLPs of H5A/indonesia/5/05 with the antigenic characteristics of H1A/brisban/59/2007, a chimeric hemagglutinin comprising the H1A/brisban/59/2007 domain fused to the H5A/indonesia/5/05 stem domain cluster was designed. The expression cassette for expressing the H5/H1 hemagglutinin fusion protein is shown in FIG. 1, and the amino acid sequence of the resulting mature fusion protein is shown in FIG. 2.

To compare the level of accumulation of the H5/H1 chimeric hemagglutinin with its native form, Nicotiana benthamiana (Nicotiana benthamiana) plants were infected with AGL1/774, AGL1/691 and AGL1/690 and the leaves were harvested after an incubation period of 6 days. To determine the level of accumulation of each HA form in agrobacterium-infected leaves, proteins were extracted from infected leaf tissue and subjected to Western blot analysis with anti-HA monoclonal antibodies. A distinct band of approximately 75kDa was detected in leaf extracts infected with AGL1/690 (figure 3), corresponding in size to the uncleaved HA0 form of influenza hemagglutinin, but not detected in AGL1/774 or AGL1/691, suggesting that the level of accumulation of chimeric hemagglutinin comprising the H1A/brisbane/59/2007 receptor binding region fused to the H5A/indonesian/5/05 backbone is higher than the native form of H1A/brisbane/59/2007 (AGL1/774) and also higher than the esterase and receptor binding region associated with H1A/brisbane/59/2007 and the chimeric hemagglutinin of the H5A/indonesian/5/05 backbone. Inactivated whole virus (WIV) (H1A/brisban/59/2007) used as a positive control was detected as multiple bands, the major band at about 80kDa, corresponding to the molecular weight of HA0, the H1A/brisban/59/2007 precursor. These results demonstrate that replacement of the corresponding portion of H5A/indonesia/5/05 with the receptor binding region of H1A/brisban/59/2007 produces a chimeric hemagglutinin that displays the antigenic region of H1 and which accumulates at higher levels in plants than native H1A/brisban/59/2007. However, chimeric hemagglutinin in which the esterase and receptor binding regions in H5A/Indonesia/5/05 were replaced with the corresponding portions of H1A/Brisban/59/2007 did not accumulate to detectable levels in plants.

Fusion of the receptor binding region of H1A/brisban/59/2007 with the H5A/indonesia/5/05 backbone as a means of increasing the cumulative levels of VLPs presenting H1 antigen in plants was reevaluated under the control of a strong CPMV-HT-based expression cassette. This fusion strategy was also compared to a signal peptide replacement approach that increased the level of accumulation. The expression cassette for expressing the H5/H1 hemagglutinin fusion protein under the control of CPMV-HT is shown in FIG. 8, and the amino acid sequence of the resulting mature fusion protein is shown in FIG. 2.

The Nicotiana benthamiana plants were infested with AGL1/732, AGL1/733, or AGL1/734 and tobacco leaves were harvested after a6 day incubation period. To determine the level of accumulation of each HA form in agrobacterium-infected leaves, proteins were first extracted from infected leaf tissue and subjected to Western blot analysis with anti-H1 (brisban) polyclonal antibodies. A distinct band of approximately 75kDa was detected in leaf extracts infected with AGL1/732, AGL1/733 and AGL1/734 (FIG. 6), corresponding in size to the uncleaved HA0 form of influenza hemagglutinin, but, although hemagglutinin was detected in all extracts analyzed, significant differences in the level of accumulation were still observed. Under these conditions, H1A/Brilliban/59/2007 expression was barely detectable using its native signal peptide (732), and replacement of the signal peptide with the signal peptide of PDI resulted in high accumulation levels of mature H1A/Brilliban/59/2007 (733), with the highest accumulation levels of chimeric H5/H1 hemagglutinin (734). Taken together, these results indicate that fusing the receptor binding domain of H1 to the H5 backbone results in high accumulation of hemagglutinin presenting the H1 antigen, and that the level of accumulation of these fusion proteins in plants is higher than that obtained with the native form, with or without signal peptide substitution.

Example 3: the corresponding part of H1A/neokaridonia/20/99 was replaced with the Receptor Binding (RB) subdomain of H5A/indonesia/5/05. Comparison of expression of chimeric and native forms.

The use of the H1 backbone (from a/neocarlidonia/20/99) in the presentation of the H5 antigenic region was also evaluated. The expression cassette for expressing the H1/H5 hemagglutinin fusion protein is shown in FIG. 1, and the amino acid sequence of the resulting mature fusion protein is shown in FIG. 2.

To compare the level of accumulation of the H1/H5 chimeric hemagglutinin with its native form, Nicotiana benthamiana plants were infected with AGL1/660 and AGL1/696, and tobacco leaves were harvested after an incubation period of 6 days. To determine the level of accumulation of each HA form in agrobacterium-infected leaves, Western blot analysis was performed with anti-H5 (indonesia) polyclonal antibody extracted from infected leaf tissue. A distinct band of approximately 75kDa was detected in leaf extracts infected with AGL1/660 and AGL1/696 (FIG. 7), corresponding in size to the uncleaved HA0 form of influenza hemagglutinin, indicating that both native H5A/Indonesia/5/05 and H1/H5 chimeric hemagglutinin accumulated at high levels in plants.

Example 4: the extracellular domain of H5A/indonesia/5/05 was replaced with H3 or the corresponding part of influenza B. Comparison of expression of chimeras and native forms.

The following strategies for presenting the H3 and B-type toxin hemagglutinin antigen regions while increasing their accumulation in plants were evaluated: the extracellular domain of H3A/British/10/2007 or B Florida/4/2006 was fused to the transmembrane and cytoplasmic subdomains from H5A/Indonesia/5/05. Expression cassettes for the H5/H3 and H5/B hemagglutinin fusion proteins are shown in FIG. 10, and the amino acids at the fusion boundaries are shown in FIG. 11.

The levels of accumulation of H5/B chimeric hemagglutinin (745) and native HA B (739) in Nicotiana benthamiana plants were compared. Plants were infested with AGL1/739 and AGL1/745 and tobacco leaves were harvested after an incubation period of 6 days. To determine the level of accumulation of each HA form in agrobacterium-infected leaves, proteins were first extracted from infected leaf tissue and subjected to Western blot analysis with anti-B (florida) polyclonal antibodies. A distinct band of approximately 75kDa was detected in the leaf extract of one plant infected with AGL1/739 (FIG. 14), corresponding in size to the uncleaved HA0 form of influenza hemagglutinin, whereas 3 plants infected with AGL1/745 showed a corresponding hemagglutinin positive signal, indicating that the H5/B chimeric form of hemagglutinin more often achieved high accumulation levels than the native form of B hemagglutinin.

Similarly, the levels of accumulation of the H5/H3 chimeric hemagglutinin (737) and its native form (736) in Nicotiana benthamiana were compared. Plants were infested with AGL1/736 and AGL1/737 and tobacco leaves were harvested after an incubation period of 6 days. To determine the level of accumulation of each HA form in agrobacterium-infected leaves, proteinopathies were extracted from infected leaf tissue and subjected to Western blot analysis with anti-H3 (brisban) polyclonal antibodies. A distinct band of approximately 75kDa was detected in leaf extracts infected with AGL1/736 and AGL1/737 (FIG. 15), corresponding in size to the uncleaved HA0 form of influenza hemagglutinin. This result indicates that fusion of the transmembrane and cytoplasmic subdomains from H5A/indonesia/5/05 to the H3A/brisban/10/2007 extracellular domain results in a chimeric hemagglutinin with similar levels of native H3A/brisban/10/2007 accumulation.

Volume exclusion chromatography was used to assess VLP production from H5/B chimeric hemagglutinin (construct No. 745) expression. On Sephacryl by Size Exclusion Chromatography (SEC)^TMConcentrated protein extracts (1.5mL) from AGL1/745 infected plants were fractionated on an S-500HR column (GEHealthcare Bio-Science Corp., Piscataway, N.J., USA). As shown in fig. 16, a blue dextran (2M Da) elution peak appears as early as in fraction 8. The proteins in 200 μ L of each SEC eluted fraction were concentrated (5 fold) by acetone precipitation and analyzed by Western blot with anti-B (florida) polyclonal antibody (fig. 16), and chimeric hemagglutinin was predominantly present in fraction 7, indicating that HA was integrated into a high molecular weight structure. Without wishing to be bound by theory, this suggests that the chimeric HA proteins assemble into large superstructures or that they adhere to high molecular weight structures. The results obtained indicate that the chimeric HA composed of the extracellular domain of HA B Florida/4/2006 and the transmembrane and cytoplasmic subdomains of H5A/Indonesia/5/05 assembles into high molecular weight particles and that these high molecules are assembled into particles of high molecular weightThe elution profile of the quantum particles is different from that of influenza VLPs.

Example 5: H5/B chimeric hemagglutinin (construct 747; comprising B/Flo HDC and SDC fused to H5/Indo TDC) was co-expressed with Hsp70 and Hsp40 and combined with signal peptide modifications.

Expression of Hsp40 and Hsp70 in plants and co-expression with influenza hemagglutinin is described in co-pending application PCT/CA 2009/000032. Plant-derived cytoplasmic Hsp70 and Hsp40 (construct No. R870) can be co-expressed with the chimeric hemagglutinin to increase their level of accumulation in plants. Co-expression was performed by infecting Nicotiana benthamiana plants with Agrobacterium suspension containing a mixture (1: 1) of AGL1 and AGL1/R870 and AGL1/35ShcPro expressing the expression cassette for the desired chimeric HA.

Cumulative levels of H5/B chimeric hemagglutinin (B/Flo HDC and SDC fused to H5/Indo TDC) co-expressed with HSP40 and HSP70 in Nicotiana semperna plants were evaluated. Plants were infested with AGL1/747, AGL1/747+ AGL1/443 (empty vector) or AGL1/747+ AGL1/R870(HSP40/HSP70), and tobacco leaves were harvested after an incubation period of 6 days. To determine the level of accumulation of H5/B chimeric HA in Agrobacterium-infected leaves, proteins were first extracted from infected leaf tissue and analyzed by Western blot with anti-B (Florida) polyclonal antibodies. A distinct band of approximately 75kDa was detected in leaf extracts of 3 plants infected with AGL1/747+ AGL1/R870 (FIG. 50), corresponding in size to the uncleaved HA0 form of influenza hemagglutinin, whereas 3 plants infected with AGL1/747+ control vector (443) showed no signal (under the exposure conditions used), indicating a high level of accumulation of hemagglutinin in the H5/B chimeric form upon co-expression with HSP40 and HSP70 chaperone proteins.

All cited documents are incorporated by reference herein as if each specific document were specifically and individually indicated to be incorporated by reference herein and as if fully set forth herein. Citation of references herein is not to be construed or as an admission that such references are prior art to the present invention.

Certain terms are used extensively in the specification and are provided with definitions to aid in understanding aspects of the invention. The examples (including examples of terms) used in the specification are for illustrative purposes only and are not intended to limit the scope and meaning of the embodiments of the present invention. Numerical ranges include the numbers defining the range. In the specification, the word "comprise" is used as an open-ended term, substantially equivalent to the term "including, but not limited to," and the word "comprising" has a corresponding meaning.

One or more presently preferred embodiments of the present invention have been described by way of example. The invention includes all embodiments, modifications and variations substantially as hereinbefore described and with reference to the examples and figures. It will be apparent to those skilled in the art that certain changes and modifications may be made without departing from the scope of the invention as defined in the following claims. Examples of such modifications include the substitution of known equivalents for any aspect of the invention in substantially the same way to achieve the same result.

Claims

1. A nucleic acid comprising one or more regulatory regions effective in a plant, insect, or yeast cell, and operably linked to a sequence encoding a chimeric influenza HA polypeptide comprising a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC), and a Transmembrane Domain Cluster (TDC), the one or more regulatory regions further comprising a 5 'UTR, a 3' UTR, or a 5 'UTR and a 3' UTR effective in the plant, insect, or yeast cell, and wherein:

a) the SDC comprises F '1, F' 2, and F subdomains;

b) the HDC comprises RB, E1, and E2 subdomains;

c) the TDC comprises TmD and Ctail subdomain; and also

Wherein at least the RB subdomain is from a first influenza HA and the SDC, TDC, or both are from one or more second influenza HA, wherein the E1, E2, or both subdomains and F '1, F' 2, or both subdomains are from the same influenza HA.

2. The nucleic acid of claim 1, wherein the sequence encoding the chimeric influenza HA polypeptide further comprises a signal peptide sequence selected from the group consisting of: HA native signal peptide sequence, alfalfa PDI signal peptide sequence, influenza H5 signal peptide sequence, and influenza H1 signal peptide sequence.

3. The nucleic acid of claim 1, wherein the 5 'UTR, 3' UTR, or 5 'UTR and 3' UTR are obtained from a plastocyanin UTR or CPMV UTR.

4. The nucleic acid of claim 1, wherein the regulatory region is obtained from a plastocyanin regulatory region, a ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) regulatory region, a chlorophyll a/b binding protein (CAB) regulatory region, a CaMV35S regulatory region, an actin regulatory region, an ubiquitin regulatory region, a triosephosphate isomerase 1 regulatory region, a translation initiation factor 4A regulatory region, and an ST-LS1 regulatory region.

5. The nucleic acid of claim 1, wherein the first and second influenza HA are independently selected from the group comprising H1, H3, H5, and B.

6. A method of producing chimeric influenza Virus Like Particles (VLPs) in a plant, comprising:

a) introducing into said plant or part thereof a nucleic acid according to claim 1, and

7. The method of claim 6, wherein in the step of introducing (step a), the nucleic acid is introduced into the plant in a transient manner.

8. The method of claim 6, wherein in the step of introducing (step a), the nucleic acid is introduced into the plant such that the nucleic acid is stably integrated into the genome.

9. The method of claim 6, further comprising the step of:

c) harvesting the plant and purifying the VLP.

10. A polypeptide encoded by the nucleic acid of claim 1.

11. A virus-like particle (VLP) comprising the polypeptide of claim 10.

12. The VLP of claim 11, further comprising plant-specific N-glycans or modified N-glycans.

13. A composition comprising an effective dose of the VLP of claim 12 and a pharmaceutically acceptable carrier.

14. Use of the virus-like particle of claim 11 in the manufacture of a medicament for inducing immunity to influenza virus infection in a subject.

15. The use of claim 14, wherein the virus-like particle is suitable for oral, intradermal, intranasal, intramuscular, intraperitoneal, intravenous, or subcutaneous administration to a subject.

16. The nucleic acid of claim 1, wherein the RB subdomain is from a first influenza HA and the E1, E2, SDC, TDC, or combination thereof is from a second influenza HA.

17. A polypeptide encoded by the nucleic acid of claim 16.

18. A virus-like particle (VLP) comprising the polypeptide of claim 17.

19. A method of producing chimeric influenza Virus Like Particles (VLPs) in a plant, comprising:

a) introducing into said plant or part thereof a nucleic acid according to claim 16, and

20. The nucleic acid of claim 1, wherein the SDC and HDC are from a first influenza HA and the TDC is from a second influenza HA.

21. A polypeptide encoded by the nucleic acid of claim 20.

22. A virus-like particle (VLP) comprising the polypeptide of claim 21.

23. A method of producing chimeric influenza Virus Like Particles (VLPs) in a plant, comprising:

a) introducing into said plant or part thereof a nucleic acid according to claim 20, and

24. A nucleic acid comprising one or more regulatory regions operably linked to a sequence encoding a chimeric influenza HA polypeptide comprising a Stem Domain Cluster (SDC), a Head Domain Cluster (HDC), and a Transmembrane Domain Cluster (TDC), wherein:

a) the SDC comprises F '1, F' 2, and F subdomains;

b) the HDC comprises RB, E1, and E2 subdomains;

c) the TDC comprises TmD and Ctail subdomain; and also

Wherein the RB subdomain is from a first influenza HA and the E1, E2, SDC, TDC, or a combination thereof is from one or more second influenza HA.

25. The nucleic acid of claim 24, wherein the E1, E2, or both subdomains and the F '1, F' 2, or both subdomains are from the same influenza HA.

26. A polypeptide encoded by the nucleic acid of claim 24.

27. A virus-like particle (VLP) comprising the polypeptide of claim 26.

28. A method of producing chimeric influenza Virus Like Particles (VLPs) in a plant, comprising:

a) introducing into said plant or part thereof a nucleic acid according to claim 24, and