BRPI0813724A2 - flu virus-like particles (vlps) comprising hemagglutinin produced inside a plant - Google Patents

Abstract

PARTICLES LIKE FLU VIRUS (VLPS) UNDERSTANDING HEMAGGLUTININ PRODUCED INSIDE A PLANT The present invention relates to a method for synthesizing flu virus-like particles (VLPs) within a plant, or a portion thereof is provided. The method involves expression of influenza HA in plants, and purification by size exclusion chromatography. The invention is also directed to a VLP comprising influenza HA protein and plant lipids. The invention is also directed to a nucleic acid encoding influenza HA, as well as vectors. VLPS can be used to formulate flu vaccines, or they can be used to enrich existing vaccines.

Description

. Patent Descriptive Report for "PARTICLES LIKE FLU VIRUS (VLPS) UNDERSTANDING HE-MAGLUTININ PRODUCED INSIDE A PLANT".

FIELD OF THE INVENTION The present invention relates to the production of virus-like particles. More specifically, the present invention is directed to the production of virus-like particles comprising influenza antigens.

BACKGROUND OF THE INVENTION Influenza is the leading cause of death in humans due to a respiratory virus. Common symptoms include fever, sore throat, shortness of breath, and sore muscle, among others. During the flu season, influenza viruses infect 10-20% of the population around the world, leading to 250-500,000 deaths annually. Influenza viruses are enveloped viruses that originate from! 15 plasma membrane of infected mammalian cells. They are classified into types A, B, or C, based on the nucleoproteins and matrix protein antigens present. Influenza type A viruses can be further divided into subtypes according to the combination of hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins presented. HA 20 governs the ability of the virus to bind to and penetrate the host cell. NA removes terminal sialic acid residues from glycan chains in the host cell and viral surface proteins, which prevents viral aggregation and facilitates virus mobility. Currently, 16 HA subtypes (H1-HI6) and 9 NA subtypes (N1-N9) are recognized. Each type A flu virus has one type of HA and one type of NA glycoprotein. Generally, each subtype exhibits species specificity; for example, all subtypes HA and NA are known to infect birds, while some subtypes Hl, H2, H3, H5, H7, H9, HlO, Nl, N2, N3 and N7 have been shown to infect humans (Horimoto 2006; Suzuki 2005). Influenza viruses comprising H5, H7 and H9 are considered the most highly pathogenic forms of influenza A viruses, and are most likely to cause future pandemics. Influenza pandemics are usually caused by

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L highly transmissible and virulent flu, and can lead to high levels of illness and death globally. The emergence of new influenza A subtypes results in 4 major pandemics in the 20th century. Spanish flu, caused by the H1N1 virus, in 1918-1919, led to the deaths of about 50 million people around the world between 1917 and 1920 Currently, the risk of the emergence of a new subtype, or of transmission to humans of an endemic subtype in animals, is always present. Of particular interest is a highly virulent form of bird flu (also known as "bird flu"), outbreaks of which have been reported in several countries 10 around the world. In many cases, this bird flu can result in death rates approaching 100 ° / °, within 48 hours. The spread of avian influenza virus (H5N1), first identified in Hong Kong ~ in 1997, to other countries in Asia and Europe, had been postulated to be linked to migratory models of wild birds. ~ 15 The current method of combating influenza in humans is through annual vaccination. The vaccine is usually a combination of several strains that are predicted to be dominant strains for the incoming "flu season". The prognosis is coordinated by the World Health Organization. Generally, the number of vaccine doses produced each year is not enough to vaccinate the world population. For example, Canada and the United States obtained enough vaccine doses to immunize about a third of their population, while only 17% of the population of the European Union was able to be vaccinated. It is clear that the current global production of influenza vaccine would be insufficient in the event of a flu pandemic in the world. Even if the necessary annual production can be found somewhat in a given year, the dominant strains change from year to year, so the stock at low necessary times in the year is not practical. Large-scale economic production of an effective flu vaccine is of significant interest to both government and private industry. 30 Viral stocks for use in vaccines are produced in fertilized eggs. The virus particles are collected, and for an inactivated viral vaccine, broken down by detergent to inactivate. Attenuated vaccines

. Alive are produced from influenza viruses that have been adapted for growth at a low temperature which means that at normal body temperature, the vaccine is attenuated.

Such a vaccine is licensed in the United States of America for use in individuals 5 to 49 years of age.

Inactivated total virus vaccines are rendered harmless by inactivation with chemical agents, and they were produced in embryonic eggs or mammalian cell culture.

All of these types of vaccines show some specific advantages and disadvantages.

An advantage of vaccines derived from total viruses is the type of immunity induced by such vaccines.

In general, vaccines divided in -O produce a strong antibody response, while vaccines produced from total viruses induce both an antibody (humoral) and a cellular response.

Even though a functional antibody response is a criterion for

"Licensing that correlates with vaccine-induced protection, there is increased evidence that a T cell response is also important in influenza immunity, this may also provide better protection in old age.

In order to induce a cellular immune response, vaccines produced from total viruses were developed.

Due to the high pathogenicity of the flu strain (for example, H5N1), these vaccines are produced in BL3 + facility. For highly pathogenic influenza strains, such as H5N1, some manufacturers have modified the hemagglutinin gene sequence to reduce the pathogenicity of the flu strain and make it avirulent and more easily produced in embryonic eggs or mammalian cell culture. .

Others also use reclassified flu strains in which the genetic sequences for hemagglutinin and neuraminidase proteins are cloned into a high-low production pathogenic influenza donor strain (A / PR / 8/34; Quan FS et al. , 2007). While these methods can produce useful vaccines, they do not provide a solution to the need for high-volume, low-cost, rapid-scale vaccines needed to meet the global need in a normal year, and would almost certainly be insufficient in the event of a pandemic.

Using this reverse genetic technology, it may also be necessary to change the genetic sequence of the HA protein to make it avirulent.

For highly pathogenic influenza strains, the production of total virus vaccines requires containment procedures, or the resulting vaccines do not exactly match the genetic sequence of the circulating virus.

In the case of live attenuated vaccines, there is still a risk that the vaccine administered may recombine with an influenza virus from the host, leading to a new influenza virus.

While this method maintains the antigenic epitope and post-translational modifications, there are a number of problems with this method, including the risk of contamination due to the use of total viruses and variable yields depending on the virus strain.

Sub-optimal levels of protection may result from genetic heterogeneity in the virus due to its introduction into the eggs.

Other disadvantages include planning to obtain eggs, risks of contamination due to chemicals used in purification, and long production times.

Also, people hypersensitive to egg proteins cannot be qualified candidates for receiving the vaccine.

In the case of a pandemic, the production of divided vaccine is limited by the need to adapt the strain for growth in eggs and the variable yields achieved.

Although this technology has been used for years to produce seasonal vaccines, it can respond very hard in a reasonable time frame to a pandemic and the capacity to manufacture around the world is limited.

To prevent the use of eggs, influenza viruses have also been produced in mammalian cell culture, for example, in MDCK or PERC.6 cells, or the like.

Another approach is reverse genetics, in which viruses are produced by cell transformation with viral genes.

These methods, however, also require the use of total virus, as well as elaborate specific culture methods and environments.

Several recombinant products have been developed as candidates for recombinant flu vaccine.

These approaches have focused on the expression, production, and purification of flu type A HA and NA proteins, including expression of these proteins using baculovirus-infected insect cells (Crawford et al., 1999; Johansson, 1999), viral vectors, and DNA vaccine construct (Olsen et al., 1997). Specifics of an influenza virus infection are well known.

Briefly, the infectious cycle is initiated by fixing the HA protein of the virion surface to a cell receptor containing sialic acid (glycoproteins and glycolipids). NA protein mediates sialic acid receptor processing, and virus penetration into the cell depends on HA-dependent receptor mediated endocytosis.

At the acidic limit of internationalized endosomes containing an influenza virus, the HA protein supports conformational changes that lead to the fusion of viral and cell membranes and uncoated viruses and the release of M2-mediated Ml proteins from ribonucleoproteins ribonucleoproteins (RNPS), which migrate in the cell nucleus for viral RNA synthesis.

Antibodies to HA proteins prevent infection of viruses by neutralizing virus infectivity, so antibodies to NA proteins mediate their effect in previous steps of viral replication.

Crawford et al. (1999) describe expression of influenza HA in insect cells infected with baculovirus.

The expressed proteins are described as being able to prevent lethal influenza disease caused by influenza subtypes H5 and H7. johansson et al. (1999) teach that influenza HA and NA proteins that express baculovirus induce immune responses in animals superior to those induced by a conventional vaccine. Immunogenicity and efficacy of hemagglutinin expressed by equine influenza baculovirus was compared to a homologous DNA vaccine candidate (Olsen et al., 1997). Collectively, these data demonstrate that a high degree of protection against influenza virus challenge can be induced with recombinant HA or NA proteins, using various experimental approaches and in different animal models.

Since previous research has shown that superficial influenza glycoproteins, HA and NA, are the primary targets for inducing protective immunity against influenza viruses, and that Ml provides a conserved target for cellular immunity to influenza, a new candidate vaccine. can include these viral antigens as a macromolecular protein particle, such as virus-like particles (VLPS). Vaccine products, VLPs, offer the advantage of being more immunogenic than subunit or recombinant antigens, and are able to stimulate both humoral and cellular immune responses (Grgacic and Anderson, 2006). In addition, the particle with these flu antigens can reveal conformational epitopes that induce neutralization of antibodies to multiple strains of influenza virus.

The production of a non-infectious flu virus strain for a vaccine proposal is a way to prevent inadvertent infection.

Alternatively, virus-like particles (VLPS) as substitutes for the cultured virus were investigated.

VLPs mimic the structure of the viral capsid, but it lacks a genome, and therefore cannot replicate or provide a means for secondary infection.

Several studies have shown that recombinant virus proteins self-aggregated in VLPS in cell culture using mammalian expression plasmids or baculovirus vectors (Gomez-Puertas et al., 1999; Neumann et al., 2000; Latham and Galarza, 2001 ). Gomez-Puertas et al. (1999) describe that the efficient formation of influenza VLPs depends on the expression levels of several viral proteins.

Neumann et al. (2000) established a system based on mammalian expression plasmid for the generation of infectious flu virus-like particles entirely from cloned cDNAS.

Latham and Galarza (2001) reported the formation of influenza VLPS in insect cells infected with recombinant baculovirus by coexpressing HA, NA, Ml, and M2 genes. These studies have shown that flu virion proteins can self-aggregate under coexpression in eukaryotic cells.

Gomez-Puertas et al. (2000) teach that, in addition to hemagluinin (HA), the matrix protein (Ml) of influenza virus is essential for VLP that originates from insect cells.

However, Chen et al. (2007) teach that Ml may not be required for VLP formation, and observed that efficient Ml and VLPS release require the presence of HA and sialidase activity provided by NA.

NA cleaves sialic acids from glycoproteins on the surface of cells that produce VLPS, and releasing VLPS into the medium. 5 Quan et al 2007 teach that the VLP vaccine produced in a baculovirus (insect cell) expression system induces a protective immunity against some strains of influenza virus (A / PR8 / 34 (H1N1)). The VLPS studied by Quan were observed to originate from the plasma membrane, and were considered to be of the correct size and morphology, similar to those obtained in a mammalian system (MDCK cells). Enveloped viruses can obtain their lipid envelope when they 'originate' from the infected cell and obtain the membrane from the plasma membrane, or that of an internal organelle.

The particles of influenza virus and VLPS originate from the host's plasma membrane.

In mammalian or baculovirus cell systems, for example, influenza originates from the plasma membrane (Quan et al 2007). Only a few enveloped viruses are known to infect plants (for example, members of Topovirus and Rhabdovirus). Of the known enveloped plant viruses, they are characterized by the origin of internal membranes of the host cell, and not from the plasma membrane.

Although a small number of recombinant VLPs have been produced in plant hosts, none have been derived from the plasma membrane, raising the question of whether plasma membrane-derived VLPs, including influenza VLPS, can be produced in plants.

Current influenza VLP production technologies rely on the coexpression of multiple viral proteins, and this dependence represents a problem with these technologies, since in the case of a pandemic and annual epidemics, response time is crucial for vaccination.

A simpler VLP production system, based on the expression of only one viral protein, is desirable to speed up vaccine development.

In order to protect the world's population from influenza and prevent future pandemics, vaccine manufacturers would need to develop rapid and effective methods of producing vaccine doses. The current use of fertilized eggs to produce vaccines is insufficient, and involves a prolonged process.

SUMMARY OF THE INVENTION It is an object of the invention to provide improved influenza virus-like particles (VLPs). In accordance with the present invention there is provided a nucleic acid comprising a nucleotide sequence that encodes an antigen from an enveloped virus operably linked to an active regulatory region in a plant. The antigen may be a flu hemagglutinin (HA). The present invention also provides a method of producing influenza virus-like particles (VLPS) in a plant comprising: a) introducing a nucleic acid that encodes an antigen of an enveloped virus, for example, a hemagglutinin from influenza (HA), operatively linked to an active regulatory region in the plant, or a portion thereof, and b) incubation of the plant or a portion thereof under conditions that allow expression of the nucleic, thereby producing VLPs. The method can also understand the steps of collecting the plant and purifying or separating the VLPs from the plant tissue. The present invention includes the above method in which, in the introduction step (step a), the nucleic acid can be either transiently expressed in the plant, or stably expressed in the plant. In addition, VLPS can be purified using size exclusion chromatography. The present invention also provides a virus-like particle (VLP) comprising an influenza virus HA protein and one or more than a plant lipid. Also included in the present invention is a composition comprising an effective dose of a VLP comprising a delay

. in influenza virus HA, one or more than a plant lipid, and a pharmaceutically acceptable carrier.

The present invention also contemplates fragments or portions of HA proteins that form VLPS in a plant. 5 The VLP may comprise an HA protein of one or more than one subtype, including Hl, H2, H3, H4, H5, H6, H7, H8, H9, HlO, Hll, H12, H13, H14, H15 or H16 , or fragment or portion thereof.

Examples of subtypes comprising such HA proteins include A / New Caledonia / 20/99 (H1N1) A / Indonesia / 5/2006 (H5N1), A / chicken / New York / 1995, 10 Herring nugget / DE / 677 / 88 (H2N8), A / Texas / 32/2003, Npato silvestre / MN / 33/00, A / duck / Shanghai / 1/2000, A / northern scribble / TX / 828189/02, A / Turkey / Ontario / 6118/68 (H8N4), A / shoveler / frog / G54 / 03, A / chicken / Germany / N / 1 949 (H1ON7), Npato / england / 56 (H11 N6), A / duck / A | berta / 60/76 (H12N5), NGaivota / Mary | and / 704/77 (H13N6), NPato 15 wild (mallard) / Gurjev / 263/82, A / duck / Australia / 341/83 (H15N8), Ngaivota de black head / Sweden / 5/99 (H1 6N3), 8 / Lee / 40, C / Johannesburg / 66, NPorto Rico / 8/34 (H1N1), A / 8risbane / 59/2007 (H1N1), Nllhas Salomão 3 / 2006 (Hl Nl), A / 8risbane 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2), B / Malaysia / 2506/2004, B / Florida / 4/2006, A / Singapore / 1/57 (H2N2), 20 A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), AA Teal / Hong Kong / W312 / 97 (H6N1), A / Equine / Prague / 56 (H7N7) , A / Hong Kong / 1073/99 (H9N2)). In one aspect of the invention, the HA protein can be a H1, H2, H3, H5, H6, H7 or H9 subtype. In another aspect, the Hl 25 protein can be from A / New Caledonia / 20/99 (H1N1), NPorto RICCO / 8/34 (H1N1), A / 8risbane / 59/2007 (H1N1) strain ), or Nllhas Salomão 3/2006 (H1N1). The H3 protein can also be from the strain of A / 8risbane 10/2007 (H3N2) or A / Wisconsin / 67/2005 (H3N2). In a further aspect of the invention, the H2 protein can be from the NSingapura / 1/530 (H2N2) strain. The H5 protein can be from the strain of A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), or A / Indonesia / 5/2005. In one aspect of the invention, the H6 stem can be from the A / Teal / Hong strain

Kong / W312 / 97 (H6N1). The H7 protein can be from the NEquino / Praga / 56 (H7N7) strain. In one aspect of the invention, the H9 protein can be from the A / Hong Kong / 1073/99 (H9N2) strain. In another aspect of the invention, the HA protein may be an influenza virus that may be a type B virus, including B / Malaysia / 2506/2004 or B / Florida / 4/2006. Examples of amino acid sequences of HA proteins of subtypes H1, H2, H3, H5, H6, H7 or H9 include SEQ ID NOs: 48-59. The HA protein of influenza virus may be H5 from Indonesia.

The present invention also provides nucleic acid molecules comprising sequences that encode an HA protein.

The nucleic acid molecules may additionally comprise one or more regulatory regions operably linked to the sequence encoding an HA protein.

The nucleic acid molecules can comprise a sequence encoding an H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. In one aspect of the invention, the HA protein encoded by the nucleic acid molecule can be a subtype H1, H2, H3, H5, H6, H7 or H9. The Hl protein encoded by the nucleic acid molecule is from the strain of A / New Caledonia / 20/99 (H1N1), NPorto Rico / 8/34 (H1N1), N8risbane / 59/2007 (H1N1), or A / llhas Salomão 3 / 2006 (H1N1). In one aspect of the invention, the H3 protein encoded by the nucleic acid molecule can be from the N8risbane strain 10/2007 (H3N2), or A / Wisconsin / 67/2005 (H3N2). In a further aspect of the invention, the H2 protein encoded by the nucleic acid molecule can be from the A / Singapore / 1/57 (H2N2) strain. The H5 protein encoded by the nucleic acid molecule can also be from the strain of A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), or A / Indonesia / 5/2005. In one aspect of the invention, the H6 protein encoded by the nucleic acid molecule can be from strain A / Teal / Hong Kong / W312 / 97 (H6N1). The H7 protein encoded by the nucleic acid molecule can also be from the NEquino / Praga / 56 (H7N7) strain. In addition, the H9 protein encoded by the nucleic acid molecule can be from the A / Hong Kong / 1073/99 (H9N2) strain. Examples of sequences of nucleic acid molecules

kleic proteins encoding such HA proteins of subtypes H1, H2, H3, H5, H6, H7 or H9 include SEQ ID NOs: 36-47 and 60-73. The nucleic acid sequence can encode the Indonesian H5 influenza virus HA protein. Regulatory regions that can be operably linked to a sequence encoding an HA protein include those that are operative in a pIanta cell, an insect cell or a cell.

Such regulatory regions may include a pIastocyanin regulatory region, a Ribulose 1,5-bisphosphate carboxylase / oxygenase regulatory region (Ru8is-1O CO), a / b-binding protein chlorofill (CAB), ST-LSI, a polyhedrin regulatory region, or a gp64 regulatory region. Other regulatory regions include a 5 'RTU, 3' RTU or terminator sequences.

The plastocyanin regulatory region can be a regulatory region for piastocyanine alfalfa; the 5 'UTR, 3'UTR or terminator sequences can be alfalfa sequences.

A method of inducing an immunity to an influenza virus infection in an individual is also provided, the method comprising administering the virus-like particle comprising an influenza virus HA protein, one or more than one lipid plant, and a pharmaceutically acceptable vehicle.

The virus-like particle can be administered to an individual orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

The present invention also belongs to a virus-like particle (VLP) comprising one or more than a protein derived from a virus selected from the group consisting of Flu, Measles, Ebola, Marburg, and HIV, and one or more than a lipid derived from a non-sialylating host production cell.

The HlV protein can be p24, gp120 or gp41; the Ebola virus protein can be VP30 or VP35; the Marburg virus protein can be Gp / SGP; the protein of the Measles virus can be H-protein or F-protein.

In addition, the present invention relates to a virus-like particle (VLP) comprising an influenza virus HA protein and one or more than a host lipid.

For example, if the host is an insect, then the virus-like particle (VLP) may comprise an HA virus flu protein and one or more than an insect lipid, or if the host is a yeast, then the particle virus-like (VLP) may comprise an influenza virus HA protein and one or more than one yeast lipids.

The present invention also relates to compositions comprising VLPS of two or more strains or subtypes of influenza.

The two or more subtypes or strains can be selected from the group comprising: A / New Caledonia / 20/99 (Hl N1) A / Indonesia / 5/2006 (H5N1), A / chicken / New York / l 995 , Herring ngaivota / DE / 677/88 (H2N8), A / Texas / 32/2003, A / wild duck / MN / 33 / OO, A / duck / Shanghai / 1/2000, Northern nrabijuncoLTX / 8281 89 / 02, A / Turkey / Ontario / 6118/68 (H8N4), A / shoveler / frog / G54 / 03, Ngalinha / Germany / N / 1949 (H10N7), A / duck / england / 56 (H1 N6), A / duck / Alberta / 60/76 (H 12N5), A / Gaovota / Maryland / 704/77 (H 13N6), A / Wild duck / Gurjev / 263/82, A / duck / Australia / 341/83 (Hl 5N8) , Wild blackheaded npato / Sweden / 5/99 (H16N3), 8 / Lee / 40, C / Johannesburg / 66, NPorto Rico / 8/34 (H1N1), A / 8risbane / 59/2007 (H1N1) , A / llhas Salomão 3/2006 (H1N1), A / 8risbane 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2), B / Malaysia / 2506/2004, B / Florida / 4/2006, A / Singapore / 1/57 (H2N2), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / Hong Kong / W312 / 97 (H6N1), A / Equine / Prague / 56 (H7N7) or A / Hong Kong / 1073/99 (H9N2)). The two or more subtypes or strains of VLPS may be present in about equivalent amounts; alternately one or more subtypes or strains may be the majority of the strains or subtypes represented.

The present invention pertains to a method for inducing immunity to influenza virus infection in a target animal or organism comprising administering an effective dose of a vaccine comprising one or more than one VLP, the VLP produced using a host of non-sialiation, for example, a plant host, an insect host, or a yeast host.

The vaccine can be administered orally, intradermally, intranasally, intramuscularly, intravenously.

. ritoneally, intravenously, or subcutaneously.

The target organism can be selected from the group comprising humans, primates, horses, pigs, birds (birds), water birds, migratory birds, corn, duck, geese, poultry, chicken, camel, canine, dogs , feline, 5 cats, tiger, leopard, civet cat, mink, stone marten, ferrets, domestic animals, mice, rats, seal, whales, and the like.

The present invention provides a method for producing VLPS containing hemagglutinin (HA) from different influenza strains in a suitable host capable of producing a VLP, for example, a plant, insect, or yeast.

The VLPS that are produced in plants contain lipids of pIanta origin, VLPS produced in insect cells comprise lipids from the plasma membrane of insect cells (commonly referred to as "insect lipids"), and VLPS produced in yeast comprise lipids from the plasma membrane of yeast cells (generally referred to as "yeast liplides"). The production of VLPs in plants has several advantages over the production of these particles in insect cell culture.

Plant lipids can stimulate specific immune cells and increase the induced immune response.

Plant membranes are produced from lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and also contain glycosphingolipids that are unique to plants and some bacteria and protozoa.

Sphingolipids are unusual in that they are not PC or PE similar to glycerol esters, but preferably consist of a long chain amino alcohol that forms an amide bond to a fatty acid chain containing more than 18 carbons.

PC and PE, as well as glycosphincopeptides, can bind to CDl molecules expressed by mammalian immune cells, such as dendritic cells similar to cells presenting antigen (APCS) and macrophages and other cells including B and T lymphocytes. in the thymus and liver (Tsuji m ,. 2006). In addition, in addition to the potential adjuvant effect of the presence of plant lipids, the ability of plant N-glycans to facilitate the capture of glycoprotein antigens by cells presenting antigen (Saint-jore-Dupas, 2007) , can be advantageous for

tion of VLPS in plants. Without wishing to be bound by theory, it is anticipated that VLPs produced from plants will induce a stronger immune reaction than VLPS produced in other manufacturing systems, and that the immune reaction by these VLPS produced by pIanta will be stronger when compared to immune reaction induced by live or attenuated total virus vaccines. Contrary to vaccines produced from total viruses, VLPS provide the advantage, since they are non-infectious, restrictive biological containment is not, therefore, a significant emission as it would be operating as a total infectious virus, and not is required for production. VLPS produced from plants provide an additional advantage again by allowing the expression system to be grown in a greenhouse or field, thus being significantly more economical and suitable for scale. Additionally, plants do not understand the enzymes involved in synthesizing and adding sialic acid residues to proteins. VLPS can be produced in the absence of neuraminidase (NA), and there is no need to coexpress NA, or treat production or extract cells with sialidase (neuraminidase), to ensure VLP production in plants. The VLPS produced in accordance with the present invention do not comprise M1 protein which is known to bind RNA. RNA is a contaminant in the VLP preparation and is undesirable when regulatory approval is obtained for the VLP product. This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features of the invention will become more similar from the following description in which reference is made to the attached drawings, in which: Figure 1A shows a sequence of an expression cassette based on plastocyanine alfalfa used for the expression of Hl according to an embodiment of the present invention (SEQ ID NO: 8). The protein disulfide isomerase (PD1) signal peptide is underlined.

The Bglll (AGATCT) and Sacl (GAGCTC) restriction sites used for cloning are shown in bold.

Figure 1B shows a schematic diagram of functional hemagglutinin domains of influenza.

After cleavage of HAO, HAI and HA2, fragments remain linked together by a disulfide bridge.

Figure 2A shows a representation of aggregated plasmid 540 for the expression of HA subtype H1.

Figure 2B shows a representation of plasmid 660 aggregated for expression of HA subtype H5. Figure 3 shows a size exclusion chromatography of leaf protein extracts producing H1 or H5 hemagglutinin. Figure 3A shows the Hl elution profile; Blue Dextran 2000 (triangles) and proteins (diamonds). Figure 3B shows immunodetection (western blot; anti HI) of H1 elution fractions following size exclusion chromatography (S50OHR beads). Figure 3C shows the H5 elution profile; Blue Dextran 2000 (triangles) and stalls (diamonds). Figure 3D shows immunodetection (western blot; anti H5) of H5 elution fractions following size exclusion chromatography (S50OHR beads). Figure 4 shows a microscopic electron photomicrograph of large H1 and H5 hemagglutinin structures from the elution fraction 9 of a size exclusion column.

Figure 4A shows a 50,000-fold magnification of an Hl VLP showing the presence of multiple similar structures (the bar represents 200 nm). Figure 4B shows a 150,000-fold magnification of an H1 VLP (the bar represents 100 nm). Figure 4C shows a 50,000-fold magnification of an H5 VLP showing the presence of multiple similar structures (the bar represents 50 nm). Figure 5A shows the sequence of the H1 Terminal-N fragment (SEQ ID NO: 1). Figure 5B shows the C-Terminal fragment of H1 (SEQ ID NO: 2). Figure 5C shows the complete H1 HAO encoding sequence (SEQ ID NO: 28). Figure 6 shows the sequence encoding H5 flanked by

© a Hindlll site immediately upstream of the initial ATG, and a Sacl site immediately downstream of the stop codon (TAA) (SEQ ID NO: 3). Figure 7A shows the sequence of the Plasto-443c primer (SEQ ID NO: 4). Figure 7B shows the SpHA (lnd) -Plasto primer sequence. 5 (SEQ ID NO: 5). Figure 7C shows the Plasto-SpHA (lnd) - (SEQ ID NO: 6) primer sequence - Figure 7D shows the HA (lnd) -Sac.r (SEQ ID NO: 7) primer sequence. Figure 8A shows the amino acid sequence of the HAI peptide sequence (SEQ ID NO: 9). Figure 8B shows the amino acid sequence of the HA5 peptide sequence (SEQ ID NO: 10). The native signal peptide is indicated in bold.

Figure 9 shows the HA sequence of influenza A subtype H7 (SEQIDNO: 11). Figure IOA shows the sequence of Influenza A HA, subtype H2 15 (SEQ ID NO: 12). Figure 108 shows the sequence of Influenza A HA subtype H3 (SEQ ID NO: 13). Figure 10OC shows the sequence of Influenza A HA subtype H4 (SEQ ID NO: 14). Figure IOD shows the sequence of Influenza A HA subtype H5 (SEQ ID NO: 15). Figure IOE shows the sequence of Influenza A HA subtype H6 (SEQ ID NO: 16). Figure IOF shows the sequence of Flu 20 A HA subtype H8 (SEQ ID NO: 17). Figure IOG shows the sequence of Group A HA subtype H9 (SEQ ID NO: 18). Figure IOH shows the sequence of Influenza A HA subtype H10 (SEQ ID NO: 19). Figure 10l shows the sequence of Influenza A HA subtype H11 (SEQ ID NO: 20). Figure 10j shows the sequence of Influenza A HA subtype H12 (SEQ ID NO: 21). Figure IOK shows the sequence of Influenza A HA subtype H13 (SEQ ID NO: 22). Figure IOL shows the sequence of Influenza A HA subtype H14 (SEQ ID NO: 23). Figure IOM shows the sequence of Influenza A HA subtype H15 (SEQ ID NO: 24). Figure ION shows the sequence of Influenza A HA subtype H16 (SEQ ID NO: 25). Figure 10O shows the sequence of Influenza B HA (SEQ ID NO: 26). Figure lOP shows the sequence of Influenza C HA (SEQ ID NO: 27). Figure IOQ shows the Xmal-pPlas.c primer sequence (SEQ ID NO: 29). Figure IOR shows the primer sequence Sacl-ATG-pPlas.r (SEQ ID NO: 30). Figure lOS

+ shows the Sacl-PlasTer.c primer sequence (SEQ ID NO: 31). Figure IOT shows the primer sequence EcoRl-PlasTer.r (SEQ ID NO: 32). Figure 11 shows a schematic representation of several constructs as used here.

Construct 660 comprises the nucleotide sequence to encode the HA subtype H5 operably linked to the plastocyanin (plast) and terminator (Pter) promoter; construct 540 comprises the nucleotide sequence to encode the HA subtype Hl in combination with an alfalfa signal peptide protein disulfide isomerase (SP PDl), and is operably linked to a plastocyanin promoter (Plast) and 10 terminator (Pter); the 544 aggregate construct for HA expression H1 subtype, the nucleotide sequence encoding H1 is combined with an alfalfa signal peptide protein disulfide isomerase (SP PDl) and a leucine zip-per GCN4pll (in place of the transmembrane domain and H1 cytoplasmic grout) and operably linked to the plastocyanin promoter 15 (Plasto) and terminator (Pter); and construct 750 for expression of influenza A / PR / 8/34 Ml coding region is combined with the 5'UTR tobacco etch virus (TEV), and operably linked with the 35S dual promoter and Nos. terminator.

Figure 12 shows H5 immunodetection, using anti-20 H5 antibodies (Vietnam), in protein extracts from N. benthamiana leaves transformed with construct 660 (lane 3). H5 commercial influenza A / Vietnam / 1203/2004 was used as a positive detection control (lane 1), and a protein extract from leaves transformed with an empty vector was used as a negative control (lane 2). 25 Figure 13 shows the characterization of hemagglutinin structures by size exclusion chromatography.

The separated biomass protein extract producing H5, Hl, soluble Hl, or Hl and Ml, was separated by gel filtration on S-500 HR.

Commercial Hl in the form of shells was also fractionated (Hl sheath). Figure 13 A shows elution fractions 30 analyzed for relative protein content (Relative Protein Level - a standard protein elution profile from a biomass fraction is shown). The Blue Dextran 2000 elution peak (reference standard 2

MDa) is indicated.

Figure 13B shows elution fractions analyzed for the presence of hemagglutinin by immunoblotting with anti-H5 antibodies (Vietnam) (for H5). Figure 13C shows elution fractions analyzed for anti-influenza A antibodies for Hl.

Figure 13D shows elution fractions analyzed for anti-influenza A antibodies to soluble H1.

Figure 13E shows elution fractions analyzed for anti-influenza A antibodies for H1 envelope. Figure 13F shows elution fractions analyzed for anti-influenza A antibodies for H1 + M1. Figure 14 shows the concentration of influenza H5 structures by 10 sucrose gradient centrifugation and electron microscopy examination of concentrated hemagglutinin fractions.

Figure 14A shows the characterization of sucrose density gradient centrifugation fractions.

Each fraction was analyzed for the presence of H5 by immunoblotting using anti-H5 antibodies (Vietnam) (top panel), and for its relative protein content and hemagglutination capacity (graph). Figure 14B shows electron microscopy examination of negative staining transmission of pooled fractions 17, 18 and 19 of sucrose gradient centrifugation.

The bar represents 100 nm.

Figure 15 shows purification of H5 influenza VLPS. Figure 20 15A shows the analysis of Coomassie Blue spotted protein content SDS-PAGE in the bleaching steps - lane 1, crude extract; lane 2, extract adjusted to pH 6; lane 3, heat treated extract; lane 4, DE-filtered extract; the fetuin affinity purification steps: lane 5, charge; lane 6, washing; lane 7, elution (10X concentrated). Figure 15B shows an electron microscopy of negative staining transmission electron from the purified H5 VLP sample.

The bar represents 100 nm.

Figure 15 C shows isolated H5 VLP enlarged to show details of the structure.

Figure 15D shows the H5 VLP product in a Coomassie-spotted SDS-PAGE (lane A) and Western blot (lane B) reduction using high rabbit polyclonal antibody 30 against HA of strain A / Vietnam / 1203 / 2004 (H5N1). Figure 16 shows a nucleotide sequence for Influenza A virus (A / New Caledonia / 20/99 (H1N1)) hemagglutinin (HA) gene, CDS

A ^

IY

Complete W. Gene Bank Accession No. AY289929 (SEQ ID NO: 33). Figure 17 shows a nucleotide sequence for Medicinal sativa mRNA for protein disulfide isomerase. Gene Bank Accession No. No. Zl 1499 (SEQ ID NO: 34). Figure 18 shows a nucleotide sequence 5 for influenza A virus (NPorto R1co / 8/34 (H1N1)) segment 7, complete sequence. Gene Bank Accession No. NC 002016.1 (SEQ ID NO: 35). Figure 19 shows the location of VLP accumulation by observing positive staining transmission electron microscopy of 10 H5-producing tissue. CW: cell wall, ch: chloroplast, pm: plasma membrane, VLP: virus-like particle. The bar represents 100 nm. Figure 20 shows induction of antibody serum responses 14 days after impulse in Balb / c mice vaccinated with HL group VLP flu produced by plant or soluble recombinant HA. Figure 20 (A) 15 antibody responses from mice immunized by intramuscular injection. Figure 20 (B) antibody responses of mice immunized through intranasal administration. Antibody responses were measured against total inactivated H5N1 viruses (Indonesia / 5/5). GMT: geometric medium titration. The values are the GMT (| og2) of titrations of 20 reciprocal end points of five mice per group. The bars represent average deviation. * p <0.05 compared to recombinant soluble HA. Figure 21 shows hematoglutination inhibition antibody (HAI) response 14 days after impulse in Balb / c mice vaccinated with VLP H5 produced from plant or recombinant soluble HA. Figure 25 21 (A) antibody responses of mice immunized through intramuscular injection. Figure 21 (B) Antibody responses of mice immunized through intranasal administration. HAI antibody responses were measured using total inactivated H5N1 viruses (A / Indonesia / 5/05). GMT: geometric mean titration. Values are GMT 30 (| og2) of reciprocal end point titrations of five mice per group. The bars represent average deviation. * p <0.05 and ** p "0.01 compared to recombinant soluble HA.

+ m

<Figure 22 shows the adjuvant effect on the immunogenicity of VLP mice in mice.

Figure 22 (A) effect of alum in mice immunized by intramuscular injection.

Figure 22 (B) Effect of Chitosan in mice immunized through intranasal administration.

HAI antibody responses were measured using total inactivated H5N1 viruses (A / Indonesia / 5/5). GMT: geometric mean titration.

The values are the GMT (log2) of reciprocal end point titrations of five mice per group.

The bars represent average deviation. * p <0.05 compared to the corresponding recombinant soluble HA. 10 Figure 23 shows antibody response to VLP administration.

Figure 23 (A) isotype of anti-Indonesian immunoglobulin / 5/05 in mice vaccinated with intramuscular injection, 30 days after impulse.

The values are the GMT (log2) of reciprocal end point titrations of five mice per group.

ELIS A performed using total inactivated viruses as the coating agent.

The bars represent average deviation. * p <0.05, ** p <0.001 compared to soluble HA! corresponding recombinant.

Figure 23 (B) Antibody titers against total inactivated viruses.

All groups are statistically different for negative control.

Figure 24 shows antibody titers against inactivated viruses 20 total homologues (Indonesia / 5/5), 2 weeks after first dose (week 2), 14 days after pulse (week 5) or 30 days after pulse (week 7) . GMT: geometric medium titration.

Values are the GMT (Iog2) of reciprocal endpoint titrations of five mice per group. * p <0.05 compared to recombinant soluble HA. 25 Figure 25 shows in vitro cross-reactivity of serum antibodies. (A) antibody titers against total inactivated viruses. (B) hemagglutination inhibition titrations against various total inactivated viruses.

The values are the GMT (] og2) of reciprocal end point titrations of five mice per group.

The bars represent average deviation.

All groups 30 are statistically different for negative control. * p <0.05, ** p <0.001 compared to the corresponding recombinant soluble HA.

Figure 26 shows the effectiveness of the VLP H5 produced per plant. (THE)

Survival rate of mice after challenge with 10 LD50 (4.09 x 10 'CCID50) of influenza A / Turkey / 582/06 (H5N1) strain (B) Body weight of mice immunized after challenge.

The values are the average body weight of surviving mice. 5 Figure 27 shows Origin of plant-derived influenza VLPS. (A) Polar lipid composition of purified influenza VLPS.

The lipids contained 40 µg of protein equivalent, were extruded from VLP as described, separated by HP-TLC, and compared to the migration profile of lipids isolated from highly purified tobacco plasma (PM) membrane. The abbreviations for lipid are as follows: DGDG, Tell- | actosi | diaci | g | jcero |; gluCER, glucosyl-ceramide; PA, phosphate acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; Pl, phosphatidylinositol; PS, phosphatidylserine; SG, Steril-glycoside- (B) Neutral liplde composition of purified influenza VLPS.

The lipids contained in an equivalent of 20 µg of proteins were extracted from VLP as described, separated by HP-TLC and compared to sitosterol migration. (C) Immunodetection of the proton pump of the ATPase plasma membrane marker (PMA) in purified VLPS and highly purified tobacco leaf PM (PMl) and B Y2 tobacco cells (PMbY2). Eighteen micrograms of protein were loaded in each lane.

Figure 28 shows the rotation sequence of Dralll sites for Sacl of clone 774 - nucleotide sequence of A / 8risbane / 59/2007 (H1N1) (SEQ ID NO: 36). The coding sequence is flanked by a plastocyanin regulatory region, starting with a Dralll restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 29 shows the rotation sequence of Dralll sites for Sacl of clone 775 - nucleotide sequence of Nllhas Salomão 3/2006 (H1N1) (SEQ ID NO: 37). The coding sequence is flanked by a plastocyanin regulatory region, starting with a Dralll restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 30 shows the sequence of rotation of Dralll sites for Sacl of clone 776 - nucleotide sequence of A / 8risbane 10/2007 5 (H1N1) (SEQ ID NO: 38). The coding sequence is flanked by a plastocyanin regulatory region, starting with a Dralll restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 31 shows the sequence of rotation of Dralll sites for Sacl of clone 777 - nucleotide sequence from A / Wisconsin / 67/2005 (H3N2) (SEQ ID NO: 39). The coding sequence is flanked by a plastocyanin regulatory region, starting with a Dralll restriction site at the 5 'end and a stop codon and a Sacl Iocal at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 32 shows the sequence of rotation of Dralll sites for Sacl of clone 778 - nucleotide sequence of B / Malaysia / 2506/2004 (SEQ ID NO: 40). The coding sequence is flanked by a plastocyanin regulatory region, starting with a Dralll restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 33 shows the rotation sequence of Dralll sites for Sacl of clone 779 - B / Florida / 4/2006 nucleotide sequence (SEQ ID NO: 41). The coding sequence is flanked by a plastocyanin regulatory region, starting with a Dralll restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 34 shows the sequence of rotation of Dralll sites for Sacl of clone 780 - nucleotide sequence from A / Singapore / 1/57

(H2N2) (SEQ ID NO: 42). The coding sequence is flanked by a plastocyanin regulatory region, starting with a restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined 5.

Figure 35 shows the sequence of rotation of Dralll sites for Sacl of clone 781 - nucleotide sequence of A / Anhui / 1/2005 (H5N1) (SEQ ID NO: 43). The coding sequence is flanked by a plastocyanin regulatory region, starting with a Dralll restriction site at the 5 'end of the tremor and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 36 shows the sequence of rotation of Dralll sites for Sacl of clone 782 - nucleotide sequence of A / Vietnam / 1194/2004 (H5N1) (SEQ ID NO: 44). The coding sequence is flanked by a regulatory region, starting with a restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 37 shows the rotation sequence of Dralll sites for Sacl of clone 783 - AACerceta / Hong Kong / W312 / 97 (H6N1) nucleotide sequence (SEQ ID NO: 45). The coding sequence is secured by a regulatory region, starting with a restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 38 shows the sequence of rotation of Dralll sites for Sacl of clone 784 - nucleotide sequence of A / Equine / Plague / 56 (H7N7) (SEQ ID NO: 46). The coding sequence is flanked by a regulatory region, starting with a restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. Constraint locations are underlined; ATG is in bold and underlined.

Figure 39 shows the rotation sequence of Dralll sites for Sacl of clone 785 - A / Hong Kong / 1073/99 (H9N2) nucleotide sequence (SEQ ID NO: 47). The coding sequence is flanked by a regulatory region, starting with a restriction site at the 5 'end and a stop codon and a Sacl site at the 3' end. The 5 restriction locations are underlined; ATG is in bold and underlined. Figure 40A shows the amino acid sequence (SEQ ID NO: 48) of the 774 clone translated polypeptide (A / 8risbane / 59/2007 (H1N1)). The open reading frame of clone 774 begins with the ATG indicated in Figure 28. Figure 40B shows the amino acid sequence (SEQ ID NO: 49) 10 of the translated polypeptide of clone 775 (Nllhas Salomão 3/2006 (H1N1) ). The open reading frame of clone 775 begins with the ATG shown in Figure 29. "Figure 41A shows the amino acid sequence (SEQ ID NO: 50) of the translated polypeptide from clone 776 (A / 8risbane / 10/2007 (H3N2) ) - 15 The open reading frame 776 begins with the ATG shown in Figure 30. Figure 41B shows the amino acid sequence (SEQ ID NO: 51) of the translated polypeptide of clone 777 (A / Wisconsin / 67/2005 ( H3N2)). The open reading structure of clone 777 begins with the ATG shown in Figure 31. Figure 42A shows the amino acid sequence (SEQ ID NO: 20 52) of the translated poIipeptide of clone 778 (B / Malaysia / 2506/2004) .The open reading structure of clone 778 begins with the ATG shown in Figure

32. Figure 42B shows the amino acid sequence (SEQ ID NO: 53) of the translated polypeptide from clone 779 (B / Florida / 4/2006). The open reading structure of clone 779 begins with the ATG shown in Figure 33. 25 Figure 43 A shows the amino acid sequence (SEQ ID NO: 54) of the polypeptide translated from clone 780 (A / Singapore / 1/57 (H2N2)). The open reading frame of clone 780 begins with the ATG indicated in Figure 34. Figure 43B shows the amino acid sequence (SEQ ID NO: 55) of the polypeptide translated from clone 781 (A / Anhui / 1/2005 (H5N1 )). The open reading structure of clone 781 begins with the ATG shown in Figure

35. Figure 44A shows the amino acid sequence (SEQ ID NO:

56) of the polypeptide translated from clone 782 (A / Vietnam / 1194/2004 (H5N1)). The open reading frame of clone 782 begins with the ATG shown in Figure 36. Figure 44B shows the amino acid sequence (SEQ ID NO: 57) of the translated poIipeptide from clone 783 (NCerceta / Hong-kong / W312 / 97 5 ( H6N1)). The open reading frame of clone 783 begins with the ATG shown in Figure 37. Figure 45A shows the amino acid sequence (SEQ ID NO: 58) of the translated polypeptide from clone 784 (A / Equine / Prague / 56 (H7N7 )). The open reading frame of clone 784 begins with the ATG indicated in Figure 38. Figure 45B shows the amino acid sequence (SEQ ID NO: 59) of the polypeptide translated from clone 785 (A / Hong Kong / 1073/99 (H9N2)). The open reading structure of clone 785 begins with the ATG indicated in Figure 39. Figure 46 shows immunodetection (western blot) of VLP elution fractions produced per plant, following size exclusion chromatography.

Hemagglutinin subtypes H1, H2, H5, H6 and H9 are shown.

Hemagglutinin is detected in fractions 7-14, corresponding to the elution of VLPS.

Figure 47 shows an expression "immunoblot" analysis of a series of H1 hemagglutinin from annual epidermal strains.

Ten and twenty micrograms of protein extracts were loaded in lanes 1 and 2, respectively.

Figure 48 shows an "immunoblot" analysis of a series of H5 hemagglutinin from potential pandemic strains.

Ten and twenty 25 micrograms of protein extracts were loaded in lanes 1 and 2, respectively.

Figure 49 shows an H5 immunoblot of strain A / Indonesia / 5/2005 in protein extracts from Nicotiana tabacum leaves, agro-infiltrated with AGL1 / 660. Two plants were infiltrated and 10 and 20 µg 30 of soluble protein from each plant were loaded in lanes 1 and 2, respectively.

Figure 50 shows the in vitro cross-reactivity of serum antibodies.

Hemagglutination inhibition titrations (Hl) in ferret serum, 14 days (A) after 1 "immunization and (B) after second impulse with H5 VLP of the flu produced by plant.

HAI antibody responses were measured using the following total inactivated H5N1 viruses: A / turkey / Turkey / 1/05, 5 A / Vietnam / 1194/04, A / Anhui / 5/05 and the homologous strain A / Indonesia / 5/05. Values are the GMT (| og2) of reciprocal end point titrations of five ferrules per group.

Diagonal strip - Indonesia / 6/06 (clade 2.1.3); verified - A / turkey / Turkey / 1/05 (clade 2.2); white bar - A / Viet Nam / 1194/04 (clade 1); black bar A / Anhui / 5/05. Responders are indicated.

The bars representing the average deviation.

Figure 51 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, H5 hemagglutinin coding sequence from A / Indonesia / 5/2005 (Construction # 660), alfalfa plastocyanin 3 'RTU and terminating sequences.

Figure 52 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, Hl hemagglutinin coding sequence from A / Nava Caledonia / 20/1999 (Construct # 540 ), plastocyanine alfalfa 3 'UTR and terminator sequences.

Figure 53 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, Hl hemagglutinin coding sequence from A / 8risbane / 59/2007 (construct # 774), alfalfa plastocyanin 3 'RTU and terminator sequences.

Figure 54 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, Hll hemagglutinin coding sequence from Nllhas Salohand / 3/2006 (Hl Nl) (construct # 775), 3 'RTU plastocyanin alfalfa and terminator sequences.

Figure 55 shows the nucleic acid sequence of an HA expression case comprising plastocyanin and 5 'alfalfa promoter

RTU, H2 hemagglutinin coding sequence from A / Singapore / 1/57 (H2N2) (construct # 780), plastocyanine alfalfa 3 'RTU and terminator sequences.

Figure 56 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, H5 hemagglutinin coding sequence from A / Anhui / 1/2005 (H5N1) (construct * 781), plastocyanine alfalfa 3 'UTR and terminator sequences.

Figure 57 shows the nucleic acid sequence of a HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, H5 hemagglutinin coding sequence from A / Vietnam / 1194/2004 (H5N1) (construct # 782), plastocyanine alfalfa 3 'UTR and terminator sequences.

Figure 58 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, A / Teal / Hong Kong / W312 / 97 (H6N1) H6 hemagglutinin coding sequence ( construct # 783), plastocyanine alfalfa 3 'RTU and terminating sequences.

Figure 59 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, H9 hemagglutinin coding sequence from A / Hong Kong / 1073/99 (H9N2) (construct # 785), plastocyanine alfalfa 3 'RTU and terminating sequences.

Figure 60 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, A / 8risbane / 10/2007 (H3N2) H3 hemagglutinin coding sequence, plastocyanin 3 alfalfa 'RTU and terminating sequences.

Figure 61 shows the nucleic acid sequence of an HA expression case comprising plastocyanin alfalfa promoter and 5 'UTR, H / A / Wisconsin / 67/2005 (H3N2) H3 hemagglutinin coding sequence, plastocyanine alfalfa 3 'RTU and ter-

mining companies. Figure 62 shows the nucleic acid sequence of an HA expression case comprising plastocyanin and 5 'UTR alfalfa promoter, 5 A / Equine / Prague / 56 H7 hemagglutinin coding sequence (H7N7), plastocyanine alfalfa 3 'RTU and terminating sequences. Figure 63 shows the nucleic acid sequence of an HA expression case comprising plastocyanin and 5 'UTR alfalfa promoter, B / Malaysia / 2506/2004 HA hemagglutinin coding sequence, 3' UTR plastocyanine alfalfa terminating strings. Figure 64 shows the nucleic acid sequence of an HA expression case comprising plastocyanin and 5 'UTR alfalfa promoter, B / Florida / 4/2006 HA hemagglutinin coding sequence, 3' UTR plastocyanine alfalfa and terminating sequences. Figure 65 shows a consensus amino acid sequence (SEQ ID NO: 74) for HA from A / New Caledonia / 20/99 (H1N1) (encoded by SEQ ID NO: 33), A / 8risbane / 59/2007 (H1N1 ) (SEQ ID NO: 48), Nllhas Solomão / 3/2006 (H1N1) (SEQ ID NO: 49) and SEQ ID NO: 9. Xl (position 3) is A or V; X2 (position 52) is D or N; X3 (position 90) is K or R; X4 (position 99) is Kou T; X5 (position 111) is You H; X6 (position 145) is Vou T; X7 (position 154) is E or K; X8 (position 161) is Rou K; X9 (position 181) isVou A; X10 (position 203) is D or N; X11 (position 2O5) is R or K; X12 (position 210) is T or K; X13 (position 225) is R or K; X14 (position 268) is W or R; X15 (position 283) is T or N; X16 (position 290) is E or K; X17 (position 432) is I or L; Xl8 (position 489) is either N or D. Figure 66 shows the amino acid sequence of Hl New Kaleidonia (AAP34324.1) encoded by SEQ ID NO: 33. Figure 67 shows the amino acid sequence of H1 Porto Rico (NC _ 0409878.1) coded by SEQ ID NO: 35

DETAILED DESCRIPTION The present invention relates to the production of virus-like particles.

More specifically, the present invention is directed to the production of virus-like particles comprising influenza antigens.

The following description is of a preferred embodiment.

The present invention provides a nucleic acid comprising a nucleotide sequence that encodes an antigen from an enveloped virus, for example, influenza hemagglutinin (HA), operably linked to an active regulatory region in a plant.

In addition, the present invention provides a method of producing virus-like particles (VLPS) in a plant.

The method involves introducing a nucleic acid that encodes an antigen operatively linked to an active regulatory region on the plant, or portion of the plant, and incubating the plant or a portion of the plant under conditions that allow acid expression nucleic, thereby producing the VLPS.

VLPS can be produced from influenza viruses, however, VLPS can also be produced from another virus derived from the plasma membrane including, but not limited to, Measles, Ebola, Marburg, and HlV.

The invention includes VLPs of all types of influenza viruses that can infect humans, including, for example, but not limited to, the very prevalent subtype A (H1N1) (for example, A / New Caledonia / 20/99 (H1N1) ), the sub-type Indonesia / 5/05 (H5N1) (SEQ ID NO: 60) and type B less common (for example, SEQ ID NO: 26, Figure 100), and type C (SEQ ID NO: 27 , Figure IOP), and for HAs obtained from other flu subtypes.

VLPS of other influenza subtypes are also included in the present invention, for example, A / 8risbane / 59/2007 (Hl Nl; SEQ ID NO: 48), Nllhas Saio-hand / 3/2006 (H1N1; SEQ ID NO: 49 ), A / Singapore / 1/57 (H2N2; SEQ ID NO: 54), A / Anhui / 1/2005 (H5N1; SEQ ID NO: 55), A / Vietnam / 1194/2004 (H5N1; SEQ ID NO: 56), A / Teal / Hong Kong / W312 / 97 (H6N1; SEQ ID NO: 57), A / Hong Kong / 1073/99 (H9N2; SEQ ID NO: 59), A / 8risbane / 10/2007 (H3N2 ; SEQ ID NO: 50), A / Wisconsin / 67/2005 (H3N2; SEQ ID NO: 51), A / Equine / Prague / 56 (H7N7; SEQ ID NO: 58), B / Malaysia / 2506/2004 ( SEQ ID NO: 52), or B / Florida / 4/2006 (SEQ ID NO: 53). The present invention also belongs to influenza viruses that

affect other mammals or host animals, for example, humans, primates, horses, pigs, birds, water birds, migratory birds, quail, duck, geese, domestic animals, chicken, camel, canine, dogs, feline, cats, tiger, leopard, civet cat, marten, stone marten, ferrets, 5 domesticated animals, farm animals, mice, rats, seal, bald, and the like.

Non-limiting examples of other antigens that can be expressed in plasma membrane-derived viruses include, the HlV - p24 capsid protein; gpl20, gp41 - enveloped props, the protectors in structural VP30 and VP35; Gp / SGP (a glycosylated integral membrane protein) of Filovirus, for example Ebola or Marburg, or protein H, and Paramyxovirus F protein, for example, Measles.

The invention also includes, but is not limited to, influenza-derived VLPs that obtain an envelope lipid from the plasma membrane of the cell in which the VLP proteins are expressed.

For example, if VLP is expressed in a pIanta-based system, VLP can obtain an envelope liplde from the cell's plasma membrane.

Generally, the term "lipldeo" refers to naturally occurring fat soluble molecules (Qipofllicas). The term is also used more specifically to refer to fatty acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids), as well as other metabolites or sterols containing fat-soluble sterol.

Phospholipids are a major component of all biological membranes, along with glycolipids, sterols and proteins.

Examples of phospholipids include phosphatidylethanamine, phosphatidylcholine, phosphatidylinositol, phosphatidyl serine, and the like.

Examples of sterols include zoosterols (eg, cholesterol) and phytosterols.

More than 200 phytosterols have been identified in several plant species, the most common being campesterol, stigmasterol, ergosterol, brassicasterol, delta-7-stigmasterol, delta-7-avenasterol, daunosterol, sitosterol, 24-methyl cholesterol, cholesterol or beta-sitosterol.

As one skilled in the art would understand, the lipid composition of a cell's plasma membrane may vary with the culture or growth conditions of the cell or organism from which the cell is obtained.

Cell membranes generally comprise lipid bilayers, as well as proteins for various functions.

The localized concentrations of particular lipids can be found in the lipid bilayer, referred to as 'lipid rafts'. Without wishing to be bound by theory, lipid rafts can play significant roles in endo and exocytosis, entry or egress of viruses or other infectious agents, inter-cell signal transduction, interaction with other structural components of the cell or organism , such as intracellular and extracellular matrices.

Referring to influenza virus, the term "hemagglutinin" or "HA", as used herein, refers to a glycoprotein found outside the flu virus particles.

HA is a type I homotrimeric glycoprotein membrane, generally comprising a signal peptide, an HAI domain, and an HA2 domain comprising a membrane rotation anchor site at the C-terminal and a small cytoplasmic tail (Figure IB) . Nucleotide sequences encoding HA are well known and available - see, for example, the 81oDefence Public Health base (flu virus; see URL: biohealthbase.orçj) or Nationa! Center for Biotechnology Information (see URL: ncbi.nlm.nih.çjov), both of which are incorporated by reference.

The term "homotrimer" or "homotrimer" indicates that an oligomer is formed by three molecules of HA protein.

Without wishing to be bound by theory, the HA protein is synthesized as a monomeric precursor protein (HAO) of around 75 kDa, which joins on the surface in a trimmed trimmer protein.

Before trimerization occurs, the precursor protein is cleaved at a conserved activation cleavage site (also referred to as fusion peptide) in 2 polypeptide chains, HAI and HA2 (comprising the transmembrane region), linked by a disulfide bond.

The HAI segment can be 328 amino acids in length, and the HA2 segment can be 221 amino acids in length.

Although this cleavage may be important for virus infectivity, it may not be essential for protein trimerization.

The

Seration of HA within the endoplasmic reticulum (ER) membrane of the host cell, signal peptide cleavage and glycosylation of protein are co-translational events.

The correct refolding of HA requires glycosylation of the protein and formation of 6 intra-chain disulfide bonds.

The HA trimer 5 joins within the cis- and trans-Golgi complex, the transmembrane domain playing a role in the trimerization process.

The crystal structures of HA proteins treated with bromelain, which lack the transmembrane domain, have shown a highly conserved structure against influenza strains.

It has been established that HA supports greater conformational changes during the infection process, which requires that the precursor HAO be cleaved into the 2 polypeptide chains HAI and HA2. The HA protein can be processed (that is, it comprises domains of HAI and HA2), or it can be unprocessed (that is, it comprises the domain of HAO). The present invention pertains to the use of an HA protein comprising the transmembrane domain and includes domains of HAI and HA2, for example, the HA protein can be HAO, or HA processed to comprise HAI and HA2. The HA protein can be used in the production or formation of VLPS using a plant, or plant cell expression system.

The HA of the present invention can be obtained from any subtype.

For example, HA can be subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, HlO, H11, H12, H13, H14, H15, or H16. The recombinant HA of the present invention can also comprise an amino acid sequence based on the sequence of any hemagglutinin known in the art - see, for example, the 81o Defense Public Health base (Flu Virus; see URL: biohealthbase.orq) or National Center for Biotechnology information (see URL: ncbi.nlm.nih.qov). In addition, HA can be based on the sequence of a hemagglutinin that is isolated from one or more emerging or recently identified influenza viruses.

The present invention also includes VLPS comprising SAH obtained from one or more than one flu subtype.

For example, VLPS may comprise one or more HA of the subtype H1 (encoded by SEQ ID NO: 28), H2 (encoded by SEQ ID NO: 12), H3 (encoded by SEQ ID NO: 13), H4 ( encoded by SEQ ID NO: 14), H5 (encoded by SEQ ID NO: 15), H6 (encoded by SEQ ID NO: 16), H7 (encoded by SEQ ID NO: 11), H8 (encoded by SEQ ID NO: 17), H9 (encoded by SEQ ID NO: 18), H10 (encoded by SEQ ID NO: 19), H11 (encoded by SEQ ID NO: 20), H12 (encoded by SEQ ID NO: 21), H13 ( encoded by SEQ ID NO: 27), H14 (encoded by SEQ ID NO: 23), H15 (encoded by SEQ ID NO: 24), H16 (encoded by SEQ ID NO: 25), or a combination thereof.

One or more than one HA of one or more than one flu subtype can be co-expressed within a plant or insect cell to ensure that the synthesis of one or more of an HA results in the formation of VLPs comprising a combination of SAH obtained from one or more than one flu subtype.

The selection of the SAH combination can be determined by the intended use of the vaccine prepared from the VLP.

For example, a vaccine for use in inoculating birds may comprise any combination of HA subtypes, while VLPS useful for inoculating humans may comprise subtypes of one or more than one of the H1, H2, H3, H5, H7 subtypes, H9, HlO, Nl, N2, N3 and N7. However, other HA subtype combinations can be prepared depending on the use of the material used in the inoculation.

Therefore, the present invention is directed to a VLP comprising one or more than one HA subtype.

The present invention also provides nucleic acids that encode hemagglutinins that form VLPS when expressed in plants.

HA flu proteins exhibit a range of similarities and differences with respect to molecular weight, isoelectric point, size, glycan complement and the like.

The physicochemical properties of the various hemagglutinins can be useful to allow differentiation between SAH expressed in a plant, insect cell or yeast system, and can be of particular use when more than one HA is coexpressed in a system. simple theme.

Examples of such physical and chemical properties are provided in Table 1.

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The present invention also includes nucleotide sequences SEQ ID NO: 28; SEQ ID NO: 3; SEQ ID NO: 11, which encodes HA of H1, H5 or H7, respectively, a nucleotide sequence that hybridizes under stringent hybridization conditions to SEQ ID NO: 28; SEQ ID NO: 3; SEQ 5 ID NO: 11, or a nucleotide sequence that hybridizes under stringent hybridization conditions to a complement of SEQ ID NO: 28; SEQ ID NO: 3; SEQ ID NO: 1, in which the nucleotide sequence encodes a hemagglutinin protein that when expressed forms a VLP, and that VLP induces the production of an antibody when administered to an individual.

For example, the expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP can be used to produce an antibody that is capable of binding HA, including HA, HAO, HAI, or mature HA2 from "one or more types of flu or subtypes.

VLP, when administered to an individual, induces an immune response. 15 Hybridization under stringent hybridization conditions is known in the art (see, for example, Current Protocols in Molecular Biology, Ausubel et al., Eds. 1995 and supplements; Maniatis et al., In Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook and Russell, in Molecular Cloning: A Laboratory Manual, 3 "edition 2001; 20 each of which is incorporated herein by reference). An example of such stringent hybridization conditions it can be about 16-20 hours of hybridization in 4 X SSC at 65 ° C, followed by washing in 0.1 X SSC at 65 ° C for one hour, or 2 washes in 0.1 X SSC at 65 "C each for 20 or 30 minutes. 25 Alternatively, an eXemplar stringent hybridization condition can be overnight (16-20 hours) in 50% formamide, 4 X SSC at 42 ° C, followed by washing in 0.1 X SSC at 65 ° C for one hour, or 2 washes in 0.1 X SSC at 65 ° C each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church 30 aqueous phosphate buffer (7% SDS; 0 , 5M NaP04 buffer pH 7.2; 10 mM EDTA) at 65 ° C, with 2 washes or at 50 ° C in 0.1 X SSC, 0.1% SDS for 20 or 30 minutes each , or 2 washings at 65 "C in 2 X SSC, O, 1 ° / o SDS for 20 or

30 minutes each.

In addition, the present invention includes nucleotide sequences that are characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100% or any amount between them, sequence identity, or sequence similarity, with the nucleotide sequence encoding HA of H1 (SEQ ID NO: 28), H5 (SEQ ID NO: 3) or H7 (SEQ ID NO: 11) , in which the nucleotide sequence encodes a hemagglutinin protein that when expressed forms a VLP, and that VLP induces the production of an antibody.

For example, the expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP can be used to produce an antibody that is capable of binding to HA, including HA, HAO, HAI, or HA2 matures.

VLP, when administered to an individual, induces an immune response.

Similarly, the present invention includes SAH associated with the following subtypes H1 (encoded by SEQ ID NO: 28), "H2 (encoded by SEQ ID NO: 12), H3 (encoded by SEQ ID NO: 13), H4 (encoded by SEQ ID NO: 14), H5 (encoded by SEQ ID NO: 15), H6 (encoded by SEQ ID NO: 16), H7 (encoded by SEQ ID NO: 11), H8 (encoded by SEQ ID NO: 17) , H9 (encoded by SEQ ID NO: 18), H10 (encoded by SEQ ID NO: 19), H11 (encoded by SEQ ID NO: 20), H12 (encoded by SEQ ID NO: 21), H13 (encoded by SEQ ID NO: 27), H14 (encoded by SEQ ID NO: 23), H15 (encoded by SEQ ID NO: 24), H16 (encoded by SEQ ID NO: 25); see Figures IOA to lOP), and nucleotide sequences which are characterized as having about 70 to 100 ° / o or any amount between them, 80 to 100% or any amount between them, 90-100% or any amount between them, or 95-100% or any amount among these, sequence identity with H1 (SEQ ID NO: 28), H2 (SEQ ID NO: 12), H3 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 11), H8 (SEQ ID NO: 17) , H9 (SEQ ID NO: 18), H10 (SEQ ID NO: 19), H11 (SEQ ID NO: 20), H12 (SEQ ID NO: 21), H13 (SEQ ID NO: 27), H14 (SEQ ID NO: 23), H15 (SEQ ID NO: 24), H16 (SEQ ID NO: 25), in which the nucleotide sequence encodes a hemagglutinin protein that when expressed forms a VLP, and that VLP induces the production of a antibody.

For example, expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP can be used to produce an antibody that is capable of binding to HA, including HA, 5 HAO, HAI, or HA2 matures.

VLP, when administered to an individual, induces an immune response.

An "immune response" generally refers to an adaptive immune system response.

The adaptive immune system usually comprises a humoral response, and a cell-mediated response.

The humoral response is the aspect of immunity that is mediated by secreted antibodies, produced in cells of the B lymphocyte lineage (cell B). Secreted antibodies bind to antigens on the surfaces of invading microbes (such as viruses or bacteria), which signal them for destruction.

Humoral immunity is generally used to refer to antibody production and the accompanying processes, as well as antibody-carrying functions, including Th2 cell activation and cytokine production, memory cell generation, opsonin promotion phagocytosis, elimination of pathogenesis, and the like.

The terms "modular" or "modulation" or similar refer to an increase or decrease in a particular response or parameter, as determined by any of several commonly known or used assays, some of which are exemplified here.

A cell-mediated response is an immune response that does not involve antibodies, but rather involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to an antigen.

Cell-mediated immunity is generally used to refer to some Th cell activation, Tc cell activation and T cell mediated responses.

Cell-mediated immunity is of particular importance in the response to viral infections.

For example, the induction of antigen-specific CD8 positive T lymphocytes can be measured using an ELISPOT assay; the stimulation of CD4 positive T lymphocytes can be measured using a proliferation assay

feration.

Antibody antibody titers can be quantified using an ELISA assay; Isotypes of antigen-specific and cross-reactive antibodies can also be measured using anti-isotype antibodies (for example, anti-IgG, IgA, IgE or IgM). The methods and techniques for carrying out such tests are well known in the art.

An inhibition of hemagglutination assay (H1, or HAI) can also be used to demonstrate the vaccine-induced antibody efficacy, or vaccine composition can inhibit the agglutination of red blood cells (RBC) by recombinant HA.

The hemagglutination inhibitory antibody titers of serum samples can be assessed by HAI microtiter (Aymard et al 1973). Erythrocytes of any of several species can be used - for example, horse, turkey, chicken, or similar.

This assay provides indirect information on the HA trlmer set on the surface of the VLP, confirming the correct presentation of antigenic sites in SAH.

Cross-reactivity of HAI titrations can also be used to demonstrate the effectiveness of an immune response to other strains of virus related to the vaccine subtype.

For example, the serum of an individual immunized with a vaccine composition of a first strain (for example, VLPS of A / Indonesia 5/05) can be used in an HAI assay with a second strain of total virus, or virus particles (for example, A / Vietnam / 1194/2004), and the HAI titration determined.

The presence of cytokine or levels can also be quantified.

For example, a T helper cell response (Th1 / Th2) will be characterized by measuring cells of lFN-y and IL-4 secretion using ELISA (for example, BD Biosciences OptEIA kits). Peripheral blood mononuclear cells (PBMC) or splenocytes obtained from an individual can be cultured, and the supernatant analyzed.

T lymphocytes can also be quantified by fluorescence activated cell count (FACS), using specific fluorescent labels and methods as are known in the art.

A microneutralization assay can also be conducted to characterize an immune response in an individual, see, for example, the methods of Rowe et al., 1973. Virus neutralization titrations can be obtained in several ways, including: 1) enumeration lysis plates (plate test), following fixation / staining of violet crystal cells; 2) microscopic observation of cell lysis in the culture; 3) ELISA and spectrophotometric detection of NP virus protein (correlates with host cell virus infection). Sequence identity or sequence similarity can be determined using a nucleotide sequence comparison program, as provided within DNASIS (for example, using, but not limited to, the following parameters: GAP penalty 5, # of upper diagonals 5, fixed GAP penalty 10, k-tuple 2, fluctuation gap 10, and window size 5). However, other methods of aligning sequences for comparison are well known in the art, for example, the algorithms of Smith & Waterman (1981, Adv.

Appl.

Math. 2: 482), Needleman & Wunsch (J.

Mol.

Biol. 48: 443, 1970), Pearson & Lipman (1988, Proc.

Nat'l.

Acad.

Sci.

USA 85: 2444), and by computerized implementations of these algorithms (for example, GAP, BESTFIT, FASTA, and BLAST)., Or by manual alignment and visual inspection.

The term "hemagglutinin domain" refers to a peptide comprising either the HAO domain, or the HAI and HA2 domains. The hemagglutinin domain does not include the signal peptide, trans-membrane domain, or the cytoplasmic syrup found in the naturally occurring protein.

The term "virus-like particle" (VLP), or "virus-like particles" or "VLPs" refer to structures that self-assemble and comprise structural proteins, such as influenza HA protein.

VLPS are generally morphologically and antigenically similar to virions produced in an infection, but lack sufficient genetic information to replicate and are therefore non-infectious.

In some examples, VLPS may comprise a single protein species, or more than one protein species.

For VLPs comprising more than one protein species, the protein species may be from the same virus species, or it may comprise a protein from a different species, genus, subfamily, or virus family (as designated by nomenclature 5 ICTV). In other examples, one or more of the protein species comprising a VLP can be modified from the naturally occurring sequence.

VLPS can be produced in suitable host cells including plant and insect host cells.

Following extraction from the host cell and after further isolation and purification under suitable conditions, the VLPS can be purified as intact structures.

The VLPS produced from influenza-derived proteins according to the present invention do not comprise M1 protein.

The Ml protein is known as Iigar RNA (Wakefield and Brownlee, 1989) which is a contaminant in the VLP preparation.

The presence of RNA is undesirable when regulatory approval is obtained for the VLP product, therefore, the preparation of VLP that lacks RNA can be advantageous.

The VLPS of the present invention can be produced in a host cell that is characterized by a lack of the ability to sialylate proteins, for example, lacking sialidase, such as a plant cell, an insect cell, fungus, and other organisms including sponge, celenterado, annelida, artoropoda, mollusca, nematelminto, trochelmintos, platelmintos, chaetognatha, tentaculate, chlamydia, spirochetes, gram positive bacteria, cyanobacteria, archaebacteria, as identified in glycoforum (see, for example, the URL: q | Ycoforum. qr.ip / science / word / evolution / ES-A03E.htm |). VLPS produced as described here do not typically comprise neuraminidase (NA). However, NA can be coexpressed with HA if VLPs comprising HA and NA are desired.

A VLP produced in a plant according to some aspects of the invention can be complexed with plant-derived lipids.

The VLP can comprise a HAO, HAI or HA2 peptide. Plant-derived lipids may be in the form of a lipid bilayer, and may additionally comprise an envelope surrounding the VLP.

PIanta-derived lipids may comprise lipid components of the plasma membrane of the plant where VLP is produced, including, but not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), glycosfingolipk deos, phytosterols or a combination thereof .

A lipid derived from pIanta can alternatively be referred to as a 'plant lipid'. Examples of phytosterols are known in the art, and include, for example, stigmasterol, sitosterol, 24-methyl] ster | and cholesterol - see, for example, Mongrand et al., 2004. VLPs can be assessed for structure and size by, for example, hemagglutination assay, electron microscopy, or by size exclusion chromatography.

For size exclusion chromatography, total soluble proteins can be extracted from plant tissue by a homogenization sample (Polytron) of crushed plant material frozen in extraction buffer, and insoluble material removed by centrifugation.

PEG precipitation can also be of benefit.

The soluble protein is quantified, and the extract is passed through a Sephacryí® column. Blue Dextran 2000 can be used as a calibration standard.

Following chromatography, fractions can be further analyzed by "immunoblot" to determine the complement of the fraction protein.

Without wishing to be bound by theory, the ability of HA to bind to RBC of different animals is triggered by the affinity of HA for sialic acids ot2,3 or a2,3 and the presence of these sialic acids on the surface of RBC.

Horse and poultry HA of influenza virus agglutinated red blood cells of all various species, including turkeys, chickens, ducks, guinea pigs, humans, sheep, horses and cows; so human HAs will bind to red blood cells from turkeys, chickens, ducks, guinea pigs, humans and sheep (see also T. et al., 1997, Virology, vol 227, p493-499; and Medeiros) R et al., 2001, Virology, vol 289 p.74-85). Examples of the reactivity of the HA species of different influenza strains are shown in Tables 2A and 2B.

Table 2A: Species of RBC released by HAS from selected seasonal çjripe strains.

Sazonall Cepa No Origin Horse Peru Hl A / 8risbane / 59/2007 (Hl Nl) 774 Human + ++ A / llhas Solomão / 3/2006 775 Human + ++ (H1N1) H3 A / 8risbane / 1 0/2007 (H3N2 ) 776 Human + ++ IA / Wisconsin / 67/2005 (H3N2) L 777 I Human j + I ++ BB / Malaysia / 2506/2004 778 Human + ++ B / Florida / 4/2006 779 Human + ++ Table 2B: Species of RBC linked by HAS from selected pandemic ipejpe strains 5. Pandemia Cepa No Origin Horse Peru H2 A / Singapore / 1/57 (H2N2) 780 Human + ++ H5 A / Anhui / 1/2005 (H5N1) 781 Hu-Av ++ + A / Vietnam / 1194/2004 (H5N1) 782 Hu-Av ++ + H6 i A / Teal / Hong I 783 I Ave l ++ I +, Kong / W312 / 97 (H6N1) H7 i A / Equine / Prague / 56 (H7N7) i 784 i Equine! ++ I ++ H9 IA / Hong Kong / 1073/99 I 785 I Human I ++ i + (H9N2) As used herein, a "protein" generally refers to an amino acid stem linked by a peptide bond, which it can be linked in a secondary, tertiary or quaternary structure to achieve a particular morphology. Alternatively, the terms polypeptide, peptide or 10 fragments of peptide can be used in a similar context.

A fragment or portion of a protein, fusion protein or polypeptide includes a peptide or polypeptide comprising a subset of the amino acid complement of a particular protein or polypeptide, provided that the fragment can form a VLP when expressed.

The fragment may, for example, comprise an antigenic region, a stress response inducing region, or a region comprising a functional domain of the protein or polypeptide.

The fragment may also comprise a region or domain for proteins in the same general family, or the fragment may include sufficient amino acid sequence to specifically identify the full-length protein from which it is derived.

For example, a fragment or portion may comprise from about 6 ° / 0 to about 100 ° / 0 of the length of the total length of the protein, or any amount between them, provided that the fragment can form a VLP when expressed.

For example, from about 60% to about 1000 ° / 0, from about 70 ° / 0 to about 100 ° / 0, from about 80% to about 100%, from about 9 ° / 0 to about from 100 ° / o, from about 95% to about 10 ° / o of the length of the total length of the protein, or any amount

- among these.

Alternatively, a fragment or portion can be from about 150 to about 500 amino acids, or any amount between them, depending on the HA, and it is provided that the fragment can form a VLP when expressed.

For example, a fragment can be from 150 to about 500 amino acids, or any amount between these, from about 200 to about 500 amino acids, or any amount in between, from about 250 to about 500 amino acids, or any amount between them, from about 20 300 to about 500 or any amount between them, from 350 to about 500 amino acids, or any amount between them, from about 400 to about 500 or any amount between them, from about 450 to about 500 or any amount in between, depending on the HA, and it is provided that the fragment can form a VLP when expressed.

For example, about 5, 10, 20, 30, 40 or 50 amino acids, or any amount in between, can be removed from the C-terminus, the N-terminus, both the N and C termini of an HA protein , provided that the fragment can form a VLP when expressed.

The number of amino acids in any given sequence is 30 relative to the particular sequence, however a technician can readily determine the 'equivalence' of a particular amino acid in a sequence based on the structure and / or sequence.

For example, if 6 amino acids

N-rings were removed when constructing a clone for crystallography, this would change the specific numerical identity of the amino acid (for example, relative to the total length of the protein), but would not change the relative position of the amino acid in the structure. 5 Comparisons of a sequence or sequences can be made using a BLAST algorithm (Altschul et al., 1990. J.

Mol Biol 215: 403-410). A BLAST search allows comparison of a question sequence with a specific sequence or group of sequences, or with a larger library or database (for example, GenBank or GenPept) 10 of sequences, and not only identifies sequences that display 100% of identity, but also those with lesser degrees of identity.

Nucleic acid or amino acid sequences can be compared using

- if a BLAST algorithm.

In addition, the identity between two or more sequences can be determined by aligning the sequences together and determining the ° /) identity between the sequences.

The alignment can be performed using the BLAST Algorithm, (for example, as available through Gen8ank; URL: ncbi.nlm.nih.qov / cqi-bin / BLAST / using fault parameters: Program: blastn ; Database: nr; Expect 10; filter: missing; Alignment: pair; Genetic Question codes: Standard (1)), or BLAST2 20 via EMBL URL: embl-heidelberg.de/Services/ index.html using parameters fault: Matrix BLOSUM62; Filter: missing, echofiltro: Expect: 10, cut: faita; Braided: both; Descriptions: 50, Alignments: 50; or FASTA, using fault parameters), or by manually comparing the sequences and calculating the ° / 0 of identity. The present invention describes, but is not limited to, the cloning of a nucleic acid encoding HA into a plant expression vector, and the production of influenza VLPS from the plant, suitable for vaccine production.

Examples of such nucleic acids include, for example, but are not limited to, an influenza A / New Caledonia virus / 20/99 30 (H1N1) HA (for example, SEQ ID NO: 61), an HA subtype of A / Indonesia / 5/05 (H5N1) (for example SEQ ID NO: 60), A / 8risbane / 59/2007 (H1N1) (for example SEQ ID NO: 36, 48.62), A / Islands Solomon / 3 / 2006

(H1N1) (for example SEQ ID NO: 37, 49, 63), NSingapura / 1/57 (H2N2) (for example SEQ ID NO: 42, 54, 64), A / Anhui / 1/2005 (H5N1) ( e.g. SEQ ID NO: 43, 55, 65), A / Vietnam / 1194/2004 (H5N1) (e.g. SEQ ID NO: 44, 56, 66), A / Teal / Hong Kong / W312 / 97 (H6N1) (e.g. SEQ ID 5 NO: 45, 57, 67), A / Hong Kong / 1073/99 (H9N2) (e.g. SEQ ID NO: 47, 59, 68), A / 8risbane / 10/2007 (H3N2) (for example SEQ ID NO: 38, 50, 69), A / Wisconsin / 67/2005 (H3N2) (for example SEQ ID NO: 39, 51, 70), A / Equine / Prague / 56 (H7N7) (for example SEQ ID NO: 46, 58, 71), B / Malaysia / 2506/2004 (for example SEQ ID NO: 40, 52, 72), 8 / Fk5rida / 4/2006 10 (for example SEQ ID NO: 41, 53, 73). The corresponding clone or construct numbers for these strains are provided in Table 1. Nucleic acid sequences that correspond to SEQ ID NOS: 36-47 comprise a plasto- "cyanine upstream and operably linked to the HA coding sequence for each of the types or subtypes, as shown in Figures 28-39. - 15 The nucleic acid sequences corresponding to SEQ ID NO: 60-73 comprise an HA expression cassette comprising plastocyanin alfalfa promoter and 5 'UTR, coding sequence for hemagglutinin from an HA, alfalfa plastocyanin 3 'UTR and terminator sequences, as illustrated in Figures 51-64.20 VLPS can also be used to produce reagents comprised of recombinant influenza structural proteins that self-assemble in protein structures macromolecular functional and homotypic immunogenic, including subviral flu particles and influenza VLP, in transformed host cells, for example, plant or cell cells s of 25 insect.

Therefore, the invention provides VLPS, and a method for producing viral VLPS in a plant expression system, from the expression of a simple envelope protein.

VLPs can be influenza VLPs, or VLPS produced from other plasma membrane-derived viruses including, but not limited to, Measles, Ebola, Marburg, and HlV.

Proteins from other enveloped viruses, for example, but not limited to, Filoviridae (for example, Ebola virus, Marburg virus, or similar

res), Paramixoviridae (for example, Measles virus, Mumps virus, Respiratory syncytial virus, pneumovirus, or the like), Retroviridae (for example, Human immunodeficiency virus-l, Human immunodeficiency virus-2, Virus l-virus T cell leukemia, or the like), Flaviviridae (eg, West Nile encephalitis, Dengue virus, Hepatitis C virus, yellow fever virus, or the like), Bunyaviridae (eg, Hantavlrus or similar), Coronaviridae (for example, coronavirus, SARS, or the like), as would be known to those skilled in the art, can also be used.

Non-limiting examples of antigens that can be expressed in plasma membrane-derived viruses include the HlV - p24 capsid protein; HlV glycoproteins gp120 or gp41, Filovirus proteins including VP30 or VP35 of Ebolavirus or Gp / SGP of Marburg virus or H protein or F protein of Measles paramyxovirus.

For example, HlV P24 (for example, Gen8ank reference gi: 19172948) is the protein obtained by translating and cleaving the HlV virus gag sequence (for example, GenBank reference gi: 9629357); gp 120 and gp41 of HlV are glycoproteins obtained by translation and cleavage of the gp160 protein (for example, Gen8ank reference gi: 9629363), encoded by env of the HlV virus genome.

Ebolavirus VP30 (GenPept Reference gi: 55770813) is the protein obtained by translating the vp30 sequence of the Ebolavirus genome (for example, GenBank Reference gi: 55770807); Ebolavirus VP35 (GenPept Reference gi: 55770809) is the protein obtained by translating the vp35 sequence of the Ebolavirus genome.

Marburg Virus Gp / SGP (GenPept Reference gi: 296965) is the protein obtained by translating the (sequence) of the Marburg virus genome (Gen-Bank Reference gi: 158539108). H protein (GenPept Reference gi: 9626951) is the protein of the H sequence of the Measles virus genome (Gen8ank Reference gi: 9626945); protein F (GenPept reference gi: 9626950) is the protein of the F sequence of the Measles virus genome.

However, other coated proteins can be used within the methods of the present invention as is known to one skilled in the art.

The invention, therefore, provides a nucleic acid molecule

unique comprising a sequence encoding HlV-p24, HlV-gpl20, HlV-gp41, Ebolavirus-VP30, Ebolavirus-VP35, Marburg Gp / SGP virus, Measles H virus protein or F protein.

The nucleic acid molecule can be operably linked to an active regulatory region in an insect, mild-hard or plant cell, or in a particular plant tissue.

The present invention further provides for the cloning of a nucleic acid encoding an HA, for example, but not limited to, human influenza virus A / Indonesia / 5/05 HA (H5N1) into a plant or insect expression vector (for example, example, baculovirus expression vector) and 10 production of influenza vaccine candidates or reagents comprising recombinant influenza structural proteins that self-assemble into immunogenic homotypic macromolecular protein structures, including particles

· Subviral flu and VLP flu, in transformed plant cells or transformed insect cells. . 15 The nucleic acid encoding the HA of influenza subtypes, for example, but not limited to, A / New Caledonia / 20/99 (H1N1), subtype A / Indonesia / 5/05 (H5N1), A / 8risbane / 59/2007 (H1N1), Nllhas Salohand / 3/2006 (H1N1), A / Singapore / 1/57 (H2N2), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 ( H5N1), A / Teal / Hong Kong / W312 / 97 (H6N1), A / Hong 20 Kong / 1073/99 (H9N2), A / 8risbane / 10/2007 (H3N2), A / Wisconsin / 67/2005 ( H3N2), A / Equine / Prague / 56 (H7N7), B / Malaysia / 2506/2004, B / Florida / 4/2006 can be expressed, for example, using a Baculovirus Expression System in a cell line appropriate, for example, Spo-dopterafrugiperda cells (for example, cell line Sf-9; ATCC PTA-4047). Other lines of insect can also be used.

The nucleic acid encoding HA can alternatively be expressed in a plant cell, or in a plant.

The nucleic acid encoding HA can be synthesized by reverse transcription and polymerase chain reaction (PCR) using HA RNA.

As an example, the RNA may be isolated from human influenza A / New Caledonia / 20/99 (H1N1) or human influenza A / Indonesia / 5/05 (H5N1) viruses, or other influenza viruses, for example - example, A / 8risbane / 59/2007 (H1N1), A / llhas Solomão / 3/2006 (Hl NI),

A / Singapore / 1/57 (H2N2), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / Hong Kong / W312 / 97 (H6N1), A / Hong Kong / 1073/99 (H9N2), A / Brisbane / 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2), A / Equine / Prague / 56 (H7N7), B / Malaysia / 2506/2004, 8 / Florida / 4/2006, or 5 cells infected with an influenza virus.

For reverse transcription and PCR, HA RNA specific oligonucleotide primers, for example, but not limited to, human influenza A / New Caledonia / 20/99 (H1N1) HA virus sequences or influenza virus HAO sequences human A / Indonesia / 5/05 (H5N1), or HA sequences of influenza subtypes 10 A / 8risbane / 59/2007 (H1N1), Nllhas Solomão / 3/2006 (H1N1), A / Singapore / 1/57 (H2N2 ), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / Hong Kong / W312 / 97 (H6N1), A / Hong Kong / 1073/99

- (H9N2), A / Brisbane / 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2), A / Equine / Prague / 56 (H7N7), B / Malaysia / 2506/2004, B / Florida / 4/2006 can be used 15.

In addition, a HA-encoding nucleic acid can be chemically synthesized using methods as known to one skilled in the art.

The resulting cDNA copies of these genes can be cloned into a suitable expression vector as required by the host expression system 20.

Examples of appropriate expression vectors for plants are described below, alternatively, baculovirus expression vector, for example, pFast8acl (lnVitrogen), resulting in pIasmids based on pFast8acl, using known methods, and information provided by the instructions from the manufacturer can be used. The present invention is additionally directed to a gene construction comprising a nucleic acid encoding HA, as described above, operably linked to a regulatory element that is operative in a plant.

Examples of regulatory elements operative in a plant cell and which can be used in accordance with the present invention include, but are not limited to, the plastocyanin regulatory region (US 7,125,978; which is incorporated herein by reference), or a regulatory region of Ribulose 1,5-bisphosphate carboxylase / oxygenase (Ru8isCO; US

4,962,028; which is incorporated by reference here), chlorophyll-binding protein a / b (CAB; Leutwiler et al; 1986; which is incorporated by reference), ST-LSI (associated with the oxygen emitted complex of photosystem ll and described by Stockhaus et al. 1987,1989; which is incorporated herein by reference). An example of a pIastocyanin regulatory region is a sequence comprising nucleotides 10-85 of SEQ ID NO: 36, or a similar region of any of SEQ ID NOS: from 37 to 47. If the construction is expressed in an insect cell, examples of regulatory elements operative in an insect cell include, but are not limited to, the polyhedrin promoter (Possee and Howard 1987. Nucleic Acids Research 15: 10233-10248), the gp64 promoter (Kogan et al., 1995. J Virology 69: 1452-1461) and the like.

. Therefore, an aspect of the invention provides a nucleic acid comprising a regulatory region and a sequence encoding an HA of influenza. The regulatory region can be a regulatory element for plastocyanin, and influenza AH can be selected from the group of flu strains or subtypes, comprising A / New Caledonia / 20/99 (Hl Nl), subtype A / Indonesia / 5 / 05 (H5N1), A / 8risbane / 59/2007 (H1N1), A / llhas Solomão / 3/2006 (Hl Nl), A / Singapore / 1/57 (H2N2), A / Anhui / 1/2005 20 ( H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / Hong Kong / W312 / 97 (H6N1), A / Hong Kong / 1073/99 (H9N2), A / 8risbane / 1 0/2007 (H3N2 ), A / Wisconsin / 67/2005 (H3N2), A / Equine / Prague / 56 (H7N7), B / Malaysia / 2506/2004, B / Florida / 4/2006. Nucleic acid sequences comprising a plastocyanin regulatory element and an HA of influenza 25 are exemplified here by SEQ ID NOS: from 36 to 47. It is known that there may be sequence differences in the hemagglutinin sequence of the amino acid sequences of influenza, or the nucleic acids that encode them, when influenza virus is grown in animals, or mammalian cells (for example, MDCK cells), or when isolated from an infected individual. Non-limiting examples of such differences are illustrated here, including Example 18. In addition, as one skilled in the art will understand, additional variation can be seen within the influenza hemagglutinin obtained from new strains as additional mutations continue to occur. to occur.

Due to the known sequence variability among known influenza hemagglutinins, the present invention includes VLPS that can be produced using any influenza hemagglutinin that, when expressed in a host as described here, influenza hemagglutinin forms a VLP.

Sequence alignments and consensus sequences can be determined using any of several software packages known in the art, for example, MULTALIN (F.

CORPET, 1988, Nucl. 10 Acids Res., 16 (22), 10881-10890), or sequences can be aligned manually and similarities and differences between the determined sequences.

The structure of hemagglutinins is well studied and the structures

· Are known to be highly conserved.

When hemagglutinin structures are superimposed, a high degree of structural consequence is. 15 observed (rmsd <2A). This structural conservation is observed even though the amino acid sequence may vary in some positions (see, for example, Skehel and Wiley, 2000 Ann Rev Biochem 69: 531-69; Vaccaro et al 2005). The hemagglutinin regions are also well conserved, for example: 20 · Structural domains: the polyprotein HAO is cleaved to provide mature HA.

HA is a homotrimer with each monomer comprising a receptor binding domain (HAI) and a membrane anchoring domain (HA2) linked by a single disulfide bond; the 20 termini-N residues of the HA2 subunit can also be referred to as the HA fusion domain or sequence.

A 'tail' region (inside the envelope membrane) is also present.

Each hemagglutinin comprises these regions or domains.

Individual regions or domains are typically conserved in length.

All hemagglutinins contain the same number and position of 30 intra- and intermolecular disulfide bridges.

The quality and position in the amino acid sequence of the cysteines that participate in the disulfide source network are conserved among SAH.

Examples of structures illustrating the characteristic disulfide bridges intra- and intermolecular and other conserved amino acids and their relative positions are described in, for example, Gamblin et al. 2004 (Science 303: 1838-1842). Exemplary structures and sequences include 1 RVZ, IRVX, IRVT, 1 RVO, IRUY, 1RU7, available from the Protein Database (URL: www.rcsb.orq). · Cytoplasmic tail - the majority of hemagglutinins comprises 3 cysteines in conserved positions.

One or more of these cysteines can be plamitoylated as a post-translational modification.

Amino acid variation is tolerated in influenza virus hemagglutinins.

This variation provides few strains that are continuously identified.

Infectivity among new strains can vary.

However, the formation of hemagglutinin trimer, which subsequently forms VLPS is maintained.

The present invention, therefore, provides a hemagglutinin amino acid sequence, or nucleic acid that encodes a hemagglutinin amino acid sequence, which forms VLPS in a plant, and includes known sequences and variant sequences that can develop.

Figure 65 illustrates an example of such a known variation.

This figure shows a consensus amino acid sequence (SEQ ID NO: 74) for HA of the following H1N1 strains: A / New Caledonia / 20/99 (H1N1) (encoded by SEQ ID NO: 33), A / 8risbane / 59 / 2007 (H1N1) (SEQ ID NO: 48), A / llhas Solomão / 3/2006 (H1N1) (SEQ ID NO: 49) and SEQ ID NO: 9. Xl (position 3) is A or V; X2 (position 52) is D or N; X3 (position 90) is K or R; X4 (position 99) is K or T; X5 (position 111) is Y or H; X6 (position 145) is V or T; X7 (position 154) is E or K; X8 (position 161) is Rou K; X9 (position 181) isVou A; X1O (position 203) is D or N; X11 (position 2O5) is R or K; X12 (position 210) is T or K; X13 (position 225) is R or K; X14 (position 268) is W or R; X15 (position 283) is T or N; X16 (position 290) is E or K; X17 (position 432) is I or L; X18 (position 489) is N or D.

As another example of such variation, a consensus and sequence alignment for HA of A / New Caledonia / 20/99 (H1N1) (coded by SEQ ID NO: 33), A / 8risbane / 59/2007 (H1N1 ) (SEQ

ID NO: 48), A / llhas Salomão / 3/2006 (H1N1) (SEQ ID NO: 49), A / Porto Rico / 8/34 (H1N1) and SEQ ID NO: 9 is shown below in Table 3 .

Table 3: Sequence alignment and consensus sequence for HA of selected H1N1 strains SEQ ÍD NO. Sequence 1 50 75 MKAKLLVLLC tftatyadti cigymnnst dtvdtvlbkn vrvmsvNIíb 9 MKAKLLVLLA TrrÀTY = í cígyhannst dtvdtvlekn vT \ rmsvNLib 48 VLLC TFTATYADTI CIGYHnST ttn "Ltk" Ltk "LST" kt "7tk" tatyadti çjgyhannst dtvdtvlekn vEvth & vn1l 51 IQQ 75 EDSHNGKLCL LKGIÀPLQLG NCSVAGWILG NPLISK ESWSYlVETP CL-9 EDSKNGKL LKGIAPLQLG NCSVAGWILG "NPECELLISK ESWSYIVETP 48 ENSHNGKLCL t0KGtAPlALG NCSVAGWILG NPECELLISK ESWSYIVEKP 49 EDSHNGKLCL LKGTAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKP € 7 m. «G è. Consensus * ^ bm emmgklcl lkgLap-lqlg ncsvawijg npecelíís éÉwsyive W ip 101 150 75 NPENGTCYPG YPAMÍBELRE QLSSVSSFER FEIFFKESSW FNHTVTGVSA 9 NPENGTCYPG YFAIJYEELRE QLSSVSSFER FEIFPKESSW PMTVTGVSA 4B NPENGTCYPG HFAUY6ELRB QLSSVSSFER FEIFPKESSW PNHTVTGVSA 49 NPENLPCYPG HFADYEELRE QLSSVSSFER FEIFPKESSW pNHTTmvsA 76. ,,. ++.,. , Xf & Consensus npengtcypg dYeelre QLB $ £ vs8EBr fei pkessw pnhExtgvse 151 200 75 scsEmKssF YRNLLYU, TGK NGLYPNLSKS YVNNKEKEVL VLhGVHHPFN 9 SCSSF YRNLLRTGK NGL ¥ FNLSKS YVNNKEKEVL vLi9ppN 48 SCSKNGBSSF YRNLLTK NGLYPNLSKS YANNKEKEVL VLWGVHHPPN SCSKNGBSSF ¥ 49 KNLLWL, TGK NCLYPNLSKS YANNKEKWL VLWGUKHPPN ..... 76 ., .., Consensus scshnqxssf yxnlLwltgk nglygnlsks j'xnnkekevl viwgvhhppn 250 2U1 = 75 IGNQRALYXT yvsvvss HYSRRFTPEI AKRFKVRDQE GRINYYWTLL 9 IGNQRALYHT FNAYVSVVSS HYSRRFTPEI AKRPKVKDQE GRINYYWTLL 48 IGDQKALYRT = YVSVVSS HYSRKÊTPEI AKRPKVRDQE GRÊNYYWTLL FGtlQR 49 $ ^ S LYHK ENÃYVSVV RYSRKFTPEI AKRPKVRDQE & GRFNYYWTLL 76 to b4 * .. ©. . , .... T SII PSGPLKAEIA QRLEWFAGK Consensus igxqxalyhx enayvsvvss hysrxftpe! akrPkm q # GRI. yywt1Z # 251 300 7 5 C EPGMUIFEA IAPWYA PALSRGFGSG fTTSNAPMDE! "9 XCQTPQG BPGDTIIFEA N (MLIAFWYA FALSRGFGSG IITsNApmE CDAXÇQT'PQG 48 EFGWIIF_A NMLIAP.RTA FALqSRGFGSG IINSNAFMDK CDAKCQTPQG 49 HGDTTIFEA NGNLIAPRYA FAV'RGFGSG ÍINM CDÀKCQTPQG NTDLEVL = 76.. .LKTRPIL SPLT'KG7 LGF vmjTvpsER GLQRRRFVQN Consensus #pgdt! £ iTea ngnLiapxya aL5 "rGf'g6g! # x itsnaPm çaakcqtpQg 301 350 75 ÀINSSLPFQN VHPVTIGECP KYVRSAKLRM VT.GLRNTPS IQSRGLFGAI 9 AINSSLPFQN VHPVTIGECP KYVRSAKLRM vT.gLRNrps IQSRCLFGAI 48 AINSSLPFQN VHWTIGECP KYVRSAKLRM VT.GLRNIPS IQSRGLFGAI 49 AINSSLPFQN VHFVTÍGECP KYVRSAKLRM VT.GLRNIPS IQSRGLFGAI

76 alng. . . . .N GDPNNMDKAV KLYRKLKREI TFHGAKEISL SYSAGÀLASC AíNsglpLqN vhPvzigecp K Consensus níRgaxlrm vtxGlrâ ips: ÍqSrG1 trans £ £ 351 400 75 AGFI ggmN F \ / DGhYGYHH QNEQGSGYAA DQKSTQNAÍN GITNKVNSVI 9 agfieggwtg MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GfTNKVNSVF 48 ageiwgwtg Wygyhh qneqgsgyaa dqkstqnaw GTTNKVNSVI 49 AgeI WYGYHH qneqgsgyaa DQKSTQNAIN 76 grm MGLIYNRM.GA = EVAFGL vcatceqiad SQHRSKRQEN TTTNPLIRHE ag consensus £ * 1WHtG mVdgwygkhh leqgggyAa dQkstqnain giTNkvnsv · i 401 450 75 EKMNTQFTAV GKEFNKLERR MENLNKKVDD G'FLDIYJTYNA ELLVLLENER EKMNTQFTAV GKEFNKLERR 9 'KENLNKKVDD GFLDIWTYNA ELLVLLENER

4.8 EXXNTQFTAV GKEFNKLKRR MENLNKKVDD GFIDIYTTYNA ELLVLÍLENER 49 EKMNTQFTAV GKEFNKLERR MENLNKKVDD GF'IDÍWI'YNA ELLVLL9NER 76 A .KAM6QMAGS A.VA. . . . . . .S QARQMVQÀMR Consensus ã% tqtTav gKéf # k $ er ": r mu1nkkv # dg £ xà £ wty'na fllv $ l # neR G5L 500 75 TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG CFEFYHKCNN ECMESKKNNKMNKKNGNKKNNKKNGNKKNGNKKNNKKNGNKKNKNG NLYEKVKSQL KNNAKEIGNG CFEFYKKCND ECMESVKNGT 49 TLDFHDSNVK NLYEKVKSQL KNNAXEIGNG cFEFYtüccND E = SVXNGT 76 TIGTHPSSSA GLKNDLLENL QÀYQKmgvQ mRFK -.............. Consensus TldfHàSwvk ntjy # kvks # L kkèigng cfêFyhkcnx ecmesvkngt 501 550 75 YDYPKYSEES KGNRBKIDGV KLESMGVYQI LAIYSTVASS LVLLVSLGAI 9 YDYPKYSEES KLNREKIDGV KLESMGVYQY 'AIYSTVASS LVLLVSLGAI 48 YDYPKYSEES KLNREKIDGV KLES'MGVYQI LAIYSTVASS LNLLVSLGAI 49 YDYPKYSEES KLNREKI KLESMGVYQI LArYsTvAss LVLLVSLGAL 6 7 4 r. 6 and.. 4 · .tb. VKY * VP · 9 P and q. F 7 R B' T * Consensus ydypkys klnrekiàw klesi 1aiWt'vaE $ lvlivs1.gai 551 566 75 SFWNCSNGSL QCRICI 9 sF ~ sNgm QCRLCI 48 SFWMCSNGSL QCRICI 49 SFWNCSNGSL QCRICI »6. 4. qcríci The consensus sequence indicates in the upper case letters of amino acids common to such sequences in a designated position; the lower case letters indicate amino acids common to at least half, or a majority of the sequences; the symbol ! is either of l or V; the symbol $ is any | kindaLouM; osimbo | o ° 6equa | kumumFouY; osÍmbo | o # is any one of N, D, Q, E, B or Z; the symbol "." is no amino acid (for example, a deletion); X in position 3 is either A or V; X in po- sição52équa | querumdeEouN; Xnaposição90éKouR; Xnaposição 99éTouK; Xnaposição1WéqualquerumdeYouH; Xnaposição145é Quality | querumdeVouT; Xnaposição157éKouE; Xnaposição162éRou K; Xnaposição182éVouA; Xnaposição203éNouD; Xnaposição205 éRouK; Xnaposição210éTouK; Xnaposição225éKouY; 333 Xnaposição is H or a cancellation; X at position 433 is I or L; X in position 49) is

N or D.

As another example of such variation, an alignment of consensus sequence and sequence for HA from A / Anhui / 1/2005 (H5N1) (SEQ ID NO: 55), A / Vietnam / 1194/2004 (H5N1) and A / Indonesia / 5/2006 (H5N1) (SEQ ID NO: 10) is shown below in Table 4. Table 4: Sequence alignment and consensus sequence for HA of selected H1N1 strains SEQ ID NO.

Sequence

1 5Q 10 WKXT VSLVKSDQIC IGYHANNSTE QVDTIMEKNV TVTHAQDILE 56 ~ IVLLFAT VSLVKSDQIC IGYHANNSTE QVDTTMEKNV T'VTHAQDM 55 MEKTVIiLLAt VSLVKSDQIC IGYHANNSTB QVDTLQQMtHQQMQQMQMQQHQQQMQQQQTQQTQQTQMQQTQQTTQQQTTQQM

5i 100 IO KTHNGKLCDL DGVKPÍALRD CSVÀCWL = ~ DEFINVP EWSYIVEKAN 56 KTKNGKLCDL DGVKPLILKD CSUA ~ LGN FMCDEFINVP EWSYIVEKAN 55 KT ¢ DLVKPLIL'RD CSVAGWlDnk ~ kdnKnLgDnKnkLnDnKnLGnKnLnLnnn

10l 15Q 10 ypndlcypgs najYEEu {KL lsrinkfeki qílrksswsd heassgvssa PVNDLCYPGD PNDYEEL 56, 55 KEASLGVSSA KLH LSRINHFEKI QKIPKSSWSS PANDLCYPGN FNDYEELKKL LSRINKFEKI QÍrpKssHsD HEASSGVSSA Consensus pxndlcypgx ¥ ndyeblkhl LsRINHp.ux QllPkSSWSd HEASGCNSSA

IQ 151 200 mmv ÇPYLGSPSFF RNVVWLIKKN STXPT1KKSY ZMCIHHPNDA CPYQGKSSFÊ 56 'RNVVWLIKKN STYPTIKRSY NNTNQEDLLV ~ 55 IHHFNDA CPYQGTPSFF RNVVWLIKKN NTYPTIKRSY NNTNQOLLI LWGIHHSNDA Consensus CPYqGxpSFF LB \ jTmIKKN sTYFTIKrSY nntnqedll! ly © ihhpnda

201 250 AEQTRLYQNP ID TTYISIGTST INQRLVPKIA TRSKVNGQSG RM9FFWTLLK 56 AEQTKLYQNP TTTIsvgFsr LNQRLVPRIA TRSKVNGQSG RbSEFFWTÍLK 55 AEQTKLYQNP TTYISVGTST WQRLVPKIA TRSKVNGQSG RMDFFwrILK Consensus AEQTkLY'QNp ttyis ÈgTsi 'LNQRLVpkIA trskvngqsg RM # FFwrrLK

Io 251 300 PNDAINFESN GNFIAPEYAY KÍVKKGDSA1 MKSELEYGNC NTKC * 56 ÈMGA FNDAINFESN GNFIAPEYA'Z KIVKKGDSTI MKSELEYGNC NTKCQTFMGA 55 FNDAINFESN GNFIAPEYAY KFVKKGDBM VKSEVEY = NTKçQTpIm PNljAíNFESN gnftapeyay K Consensus "IVKKGDSa1 msElEywc NTKCQTPmGA

301 350 10 INSSMPF'HNI HPLTIGECPK YVKSNRLVLÀ TGLRNSPQRE SRRKKRGWG 56 lNSSMPFHNI HPLTIGECPK YVKSNRLVLA TGLANSPQRE RRRKKRGLPG 55 INssxpFmI HPLTIGECPK YLKSRKNKLRRKNKRKKRRKNK.

RGLFG

Consensus inssmpfhnk hpltigefpk YvKSNrLVLA TGLRNSPqRE rRRKkRGLFG

Io 400 35.1 The "AGFIEGGW QGMVDGWYGY WSNEQGSGY AADKESTQKA INVTNKVNS 56 AIÀGFIEGGW QGMVDGWYGY KHSNEQGSGY AADKESTQKA IDGVTNKVNS 55 AIÀGFIEGGW GY = AADKEsmKA IDGVTNKVNS Conse NSO AIAGFIEGGW QGXVDGWYGY KHSNEQGSGY AADKÊ TQKA r $ 401 450 10 ITDKKNTQFE AvgREFlu¶jLE RRÍENLNKKM N = 56 FLDVWTY IIDKMNTQFE AVGREFNNLE RRIKM EDGFLIJVWTY NAEUÂ 55 IIDKMNTQFE Consensus aVGREFNNLE RRI.ENLNKKM EDGFLWWTY NMW i == idkmntqfe avgrefnnle rrienlnkkm naellvlmen 451 500 1U ERTLDFHDSN VKNLYDKVRL QLRDNAKELG NGCFEFYKKC DNBCMESÍRN 56 ERTLDFKDSN VKNLYDKVRL QLRDNAXELG NgcFEFYHKé DNECMESVRN 55 ERTLDFHDSN VKNLYDKVRL QLRDNAKELG NGCFEYYHKC DNECMESVRN Consensus ertldfkdsn vknlydkvrl qlrdnakelg ngcfefyhkc dnecmes irn SOI GTYNYPQYSE 550 10 55 KREEIS GVKLESIGTY QILSIYSTVA SSLALAIM GTYDYPQYSE eARLKREEIS GVKLESTGIY QFLSIYSTVA SSLALAIMVA 55 GTYDYPQYSE eARLKREEIS gVKLESIGTY QíLsIYsTvA SSLALAIMVÃ Consensus gtyvypqyse earlkreeis gvKLEsIgtY qi'lsiystva ssLALtAIMvÀ 551 568 56 lO GLSLYMCSNG SLQCRICI gLsumc8Ng slqcrici .55 GL "NG SLQCRICI Consen so glsl9mcsng sLQcRIct The consensus sequence indicates, in the upper case, letters of amino acids common to all sequences in a designated position; the lower case letters indicate amino acids common to at least half, or a majority of the sequences; the symbol ! is either I or V; the symbol $ équa | querumdeLouM; osimbo | o ° /) équa | querumdeFouY; osÍmbo | o # is any one of N, D, Q, E, B or Z; X in position 102 is any of T, V or A; X in position 11O is any S, Dou N; X in position 156 is any S, kouT. The alignments illustrated and described above and consensus sequences are non-limiting examples of variants in hemagglutinin amino acid sequences that can be used in various embodiments of the invention for the production of VLPS in a plant. A nucleic acid encoding an amino acid sequence can be easily determined, since the codons for each amino acid are known in the art. The provision of an amino acid sequence, therefore, teaches how to degenerate nucleic acid sequences that encode it. The present invention, therefore, provides a nucleic acid sequence that encodes a hemagglutinin of those flu strains and subtypes described herein (for example, A / New Caledonia / 20/99 (H1N1) A / Indonesia / 5/2006 (H5N1), A / chicken / New York / 1995, A / herring wild duck / DE / 677/88 (H2N8), A / Texas / 32/2003, Northern hemisphere wild duck / MN / 33/00, 5 A / duck / Shanghai / 1/2000, A / northern scribble / TX / 828189/02, NPeru / Ontario / 6118/68 (H8N4), A / shoveler / lrã / G54 / 03, Ngalinha / Germany / N / 1949 (H1ON7), A / duck / England / 56 (H11 N6), A / duck / Alberta / 60/76 (H12N5), N Wild duck / Maryland / 704/77 (H13N6), A / Northern hemisphere wild duck / Gurjev / 263/82 , A / duck / Australia / 341/83 10 (H15N8), A / black-headed wild duck / Sweden / 5/99 (H16N3), 8 / Lee / 40, C / Johannesburg / 66, NPorto Rico / 8/34 (Hl Nl), A / 8risbane / 59/2007 (HI Nl), A / llhas Salomão 3/2006 (H1N1), A / 8risbane 10/2007 (H3N2),

. A / Wisconsin / 67/2005 (H3N2), B / Malaysia / 2506/2004, B / Florida / 4/2006, A / Singapore / 1/57 (H2N2), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 "15 (H5N1), A / Teal / Hong-kong / W312 / 97 (H6N1), A / Equine / Prague / 56 (H7N7), A / Hong-kong / 1073/99 (H9N2) ), as well as degenerate sequences that hinder the hemagglutinins above.

In addition, an amino acid sequence encoded by a nucleic acid can be easily determined, as the codon or codon for each amino acid is known.

The provision of a nucleic acid, therefore, teaches an amino acid sequence encoded by it.

The invention, therefore, provides hemagglutinin amino acid sequences of those flu strains and subtypes described here (for example A / Nova Caxtonónia / 20/99 (H1N1) Nlndonesia / 5/2006 (H5N1), A / chicken / New York / 1995, 25 Herring wild duck / DE / 677/88 (H2N8), A / Texas / 32/2003, Northern hemisphere wild duck / MN / 33/00, A / duck / Shanghai / 1/2000 , A / northern scribble / TX / 828189/02, A / Peru / Ontario / 6118/68 (H8N4), A / shoveler / lran / G54 / 03, Nga | jnha / A | germany / N / 1949 (H10N7) , Npato / england / 56 (HHN6), A / duck / Alberta / 60/76 (H 12N5), A / Wild duck 30 / Maryland / 704/77 (HI3N6), N Northern hemisphere wild duck / Gurjev / 263 / 82, A / duck / Australia / 341/83 (H15N8), Black-necked wild duck / Sweden / 5/99 (H16N3), 8 / Lee / 40, C / Johannesburg / 66, A / Porto Ri-

CO / 8/34 (H1N1), A / Brisbane / 59/2007 (H1N1), A / llhas Solomão 3/2006 (H1N1), A / 8risbane 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2 ), B / Malaysia / 2506/2004, B / Florida / 4/2006, A / Singapore / 1/57 (H2N2), NAnhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / Hong-5 kong / W312 / 97 (H6N1), A / Equine / Prague / 56 (H7N7), A / Hong-kong / 1073/99 (H9N2)). In plants, influenza VLPS originate from the plasma membrane (see Example 5, and Figure 19); therefore, the lipid composition of the VLPS reflects its origin.

The VLPS produced in accordance with the preceding invention comprise HA of one or more than one of the flu type or subtype, complexed with plant-derived lipids.

Plant lipids can stimulate specific immune cells and increase the response

- immune induced.

Plant membranes are produced from lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and also contain glycosphingoli- pids, saponins, and phytosterols.

In addition, lipid barges are also found in plant plasma membranes — these microdomains are enriched in sphingolipids and sterols.

In plants, a variety of phytosterols are known to occur, including stigmasterol, sitosterol, 24-methyl cholesterol and cholesterol (Mongrand et al., 2004). 20 PC and PE, as well as glycosphingolipids, can bind to CDl molecules expressed by mammalian immune cells such as dendritic cells similar to cells presenting antigen (APCs) and macrophages, and other cells including B and T lymphocytes in the thymus and flank (Tsuji m, 2006). CDl molecules are structurally similar to complex molecules of greater class I histocompatibility (MHC), and their role is to present glycolipid antigens for NKT cells (Natural Killer T cells). After activation, NKT cells activate innate immune cells such as NK cells and dendritic cells, and also activate antibody similar to adaptive immune cells that produce B cells and T cells. 30 A variety of ditosterols can be found on a membrane plasma - the specific complement may vary depending on species, growth conditions, sources of nutrients or state of pathogenesis, to name a few factors.

Generally, beta-sitosterol is the most abundant phytosterol.

Phytosterols present in a flu VLP complexed with a lipid bilayer, such as a plasma membrane-derived envelope, can provide an advantageous vaccine composition.

Without wishing to be bound by theory, VLPS produced from plants complexed with a lipid bilayer, such as an envelope derived from a plasma membrane, can induce a stronger immune reaction than VLPS produced in other expression systems, and may be similar to the immune reaction 10 induced by live or attenuated total virus vaccines.

Therefore, in some embodiments, the invention provides a VLP complexed with a plant-derived lipid bilayer.

In

· In some embodiments, the plant-derived Iiplde bilayer can comprise the VLP envelope, 15 The VLP produced within a plant can induce an HA comprising plant-specific N-glycans.

Therefore, this invention also provides a VLP comprising HA having plant-specific N-glycans.

In addition, modification of N-glycan in plants is known 20 (see, for example, U.S. 60 / 944,344; which is incorporated herein by reference) and HA having modified N-glycans can be produced.

HA comprising a modified glycosylation model, for example, with reduced fucosilated, xylosylated N-glycans, or both, fucosylated and xylosylated can be obtained, or HA having a modified glycosylation model can be obtained, at which the protein lacks fucosylation, xylation, or both, and comprises increased galatosylation.

In addition, modulation of post-translational modifications, for example, the addition of terminal galactose, can result in a reduction of fucosylation and xylation of expressed HA when compared to HA of wild-type plant expression. 30 For example, which is not to be considered limiting, the synthesis of HA having a modified glycosylation model can be achieved by coexpressing the protein of interest together with a nucleotide sequence.

deo encoding beta-1.4 galactosyltransferase (GalT), for example, but not limited to mammal GalT, or human GalT however GalT from other sources can also be used.

The catalytic domain of GalT can also be fused to a CTS domain (i.e., the cytoplasmic tail, transmembrane domain, stem region) of N-acetylglucosaminyl transferase (GNTI), to produce a hybrid enzyme of GNTl-GalT, and the hybrid enzyme can be coexpressed with HA.

HA can also be coexpressed together with a nucleotide sequence that encodes N-acetylglucosaminyltrasnferase lll (GnT-lll), for example, but not limited to mammal GnT-III or human GnT-lll, GnT-III from other sources can also be used.

In addition, a hybrid enzyme from GNTl-GnT-lll, comprising the CTS of GNTI fused to GnT-lll can also be used. "Therefore, the present invention also includes VLP'S comprising HA having modified N-glycans. 15 Without wishing to be bound by theory, the presence of plant N-glycans in HA can stimulate the immune response by promoting the binding of HA by cells that present stimulus.

Stimulating the immune response using plant N-glycan was proposed by Saint-jore-Dupas et al. (2007). Furthermore, the conformation of the VLP can be advantageous for the presentation of the antigen, and increases the adjuvant effect of the VLP when complexed with a plant-derived lipid layer.

By "regulatory region" "regulatory element" or "promoter" is meant a portion of nucleic acid typically, but not always, upstream of the gene's coding region, which may be comprised of DNA or RNA, or both DNA and RNA.

When a regulatory region is active, and in operative association, or operably linked, with a gene of interest, this can result in the expression of the gene of interest.

A regulatory element may be able to mediate organ specificity, or control the activation of a developmental or temporal gene.

A "regulatory region" includes promoter elements, core promoter elements exhibiting a basal promoter activity, elements that are inducible in response to an external stimulus, elements that mediate promoter activity, such as negative or intensifying regulatory elements transcriptional.

The "regulatory region", as used herein, also includes elements that are active following transcription, for example, regulatory elements that modulate gene expression, such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activation sequences , and determinants of mRNA instability.

Several of the latter elements can be located proximal to the coding region.

In the context of this description, the term "regulatory element" or 10 "regulatory region" typically refers to a DNA sequence, usually, but not always, upstream (5 ') the coding sequence for a structural gene, which controls the expression of the coding region by predicting the recognition of RNA polymerase and / or other factors required for transcription to start a particular site.

However, it is to be understood that other nucleotide sequences, located within introns, or 3 'of the sequence, can also contribute to the regulation of expression of a coding region of interest.

An example of a regulatory element that provides for recognition of RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter element.

Many, but not all, eukaryotic promoter elements contain a TATA box, a nucleic acid sequence comprised of adenosine and thymidine base pairs usually located approximately 25 base pairs upstream of a transcriptional starting site .

A promoter element comprises a basal promoter element, responsible for initiating transcription, as well as other regulatory elements (as listed above) that modify expression expression.

There are several regulatory regions, including those that are developmentally regulated, inducible or constitutive.

A regulatory region 30 that is regulated in a developed way, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during development

that organ or tissue. However, some regulatory regions that are developmentally regulated may preferably be active within certain organs or tissues at different developmental stages, they may also be active in a developmentally regulated manner, or at a baseline level in other organs or tissues within a plant. also. Examples of tissue-specific regulatory regions, for example, see a specific regulatory region, include the napin promoter, and the cruciferin promoter (Rask et al., 1998, J. Planta Physiol. 152: 595-599; Bilodeau et al. , 1994, Plant Cell 14: 125-130). An example of a specific 10-leaf promoter includes the plastocyanin promoter (Figure 1b or SEQ ID NO: 23); US

7,125,978, which is incorporated by reference). An inducible regulatory region is one that is able to activate. directly or indirectly transcribing one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the 15 DNA sequences or genes will not be transcribed. Typically the protein factor that specifically binds to an inducible regulatory region for. activate transcription can be present in an inactive form, which is then directly or indirectly converted to the active form by the inductor. However, the protein factor may also be absent. The inducer can be a chemical agent, such as protein, metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress imposed directly by heat, cold, salt, or toxic elements, or indirectly through action pathogen or disease agent such as a virus. A plant cell containing an inducible regulatory region can be exposed to an inductor by applying the inductor externally to the cell or plant, such as spraying, irrigation, heating or similar methods. Inducible regulatory elements can be derived from either plant or non-plant genes (for example, Gatz, C. and Lenk, I.R.P., 1998, Trends Plant Sci. 3, 352-358; which is incorporated by reference). Examples of potential inducible promoters include, but are not limited to, inducible tetracycline promoter (Gatz, C., 1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108; which is incorporated by reference), inducible steroid promoter (Aoyama, T. and

Chua, N.H., 1997, Plant j. 2, 397-404; which is incorporated by reference), and inducible ethanol promoter (Salter, MG, et al., 1998, Plant Journal 16, 127-132; Caddick, MX, et al., 1998, Nature Biotech. 16, 177-180, which are incorporated by reference), cytokinin 5-inducible IB6 and CK11 genes (Brandstatter, I. and Kieber, JJ., 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985 ; which are incorporated by reference), and the inducible auxin element, DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963- 1971; which is incorporated by reference). A constitutive regulatory region directs the expression of a gene through all the various parts of a plant and continuously through the entire development of the plant.

Examples of known constitutive regulatory elements include promoters associated with the

- CaMV 35S transcript. (Odell et al., 1985, Nature, 313: 810-812), rice actin 1 (Zhang et al., 1991, Plant Cell, 3: 1155-1165), actin 2 (An et al., "15 1996, P / ant j., 10: 107-121), or tms 2 (US 5,428,147, which is incorporated herein

by reference), and triosphosphate isomerase 1 genes (Xu et. al., 1994,

. Plant Physiol. 106: 459-467), the corn ubiquitin 1 gene (Cornejo et al., 1993, Plant MoI.

Biol. 29: 637-646), Arabidopsis ubiquitin 1 and 6 genes (HOI-torf et al., 1995, Plant Mol.

Biol. 29: 637-646), and the translational tobacco initiation factor 4A (Mandel et al., 1995 Plant Mol.

Biol. 29: 995-1004). The term "constitutive", as used herein, does not necessarily indicate that a gene under control of the constitutive regulatory region is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types even though variation in abundance is frequently observed.

By "operably linked" it is significant that the particular sequences, for example, a regulatory element and a coding region of interest, interact or directly or indirectly to perform a desired function, such as mediation or modulation of expression of a gene.

The interaction of operably linked sequences can, for example, be mediated by proteins that interact with operably linked sequences.

The one or more nucleotide sequences of the present invention can be expressed in any suitable plant host that is transformed by the nucleotide sequence, or constructs, or vectors of the present invention.

Examples of suitable hosts include, but are not limited to, agricultural crops including alfalfa, canola, Brassica spp., Corn, Nicotiana spp., Alfalfa, potato, ginseng, peas, oats, rice, soy, wheat, barley, sunflower , cotton, and the like.

The one or more chimeric genetic constructs of the present invention can additionally comprise a 3 'non-translated 10' region.

A 3 'untranslated region refers to that portion of a gene comprising a segment of DNA that contains a polyadenylation signal and any other regulatory signals capable of carrying out processes.

"mRNA processing or gene expression.

The polyadenylation signal is usually characterized by effecting the addition of polyadenic acid traces to the 3 'end of the mRNA precursor.

The signs of polyadeni-. population are commonly recognized by the presence of homology for the

- canonical 5 'AATAAA-3', although variations are uncommon.

One or more of the chimeric genetic constructs of the present invention may also additionally include enhancers, or transcription or transcription enhancers, as may be required.

These enhancer regions are well known to those skilled in the art, and may include the ATG initiation codon and adjacent sequences.

The initiation codon must be in phase with the reading structure of the coding sequence to ensure translation of the total sequence. 25 Non-limiting examples of suitable 3 'regions are the untranslated 3' transcribed regions containing an Agrobacterium tumor polyadenylation signal inducing plasmid (Ti) genes, such as nopaline synthase (Nos gene) and plant genes, such as soy storage genes, a small subunit ribulose-1,5-bis-phosphate carboxyl- 30 se gene (sSRUBISCO; US 4,962,028; which is incorporated herein by reference), the promoter used in the regulation of pIastocyanin expression (Pwee and Gray 1993; which is incorporated by reference). An example of a plastocyanin promoter is described in US 7,125,978 (which is incorporated herein by reference). As described here, promoters comprising enhancer sequences with demonstrated efficiency in leaf expression have been found to be effective in transient expression.

Without wishing to be bound by theory, the fixation of regulatory elements upstream of a photosynthetic gene by fixation to the nuclear matrix can mediate strong expression.

For example, up to -784 of the translation starting location of the pea plastocyanin gene can be used to mediate strong expression of the reporting gene. To aid in the identification of transformed plant cells, the constructs of this invention can be further manipulated to include selectable plant markers.

Useful selectable markers

- include enzymes that provide resistance to chemicals such as an antibiotic, for example, gentamicin, hygromycin, kanamycin, or herbicides, such as phosphinothricin, glyphosate, chlorosulfuron, and the like.

Similarly,

"enzymes providing production of an identifiable compound by changing

. Color change such as GUS (beta-glucuronidase), or luminescence, such as luciferase or GFP, can be used.

Also considered part of this invention are transgenic plants, plant cells or seeds containing the chimeric gene construct of the present invention.

Methods of regenerating total plants from plant cells are also known in the art.

In general, transformed plant cells are grown in an appropriate medium, which may contain selective agents, such as antibiotics, where selectable markers 25 are used to facilitate identification of transformed plant cells.

Once the callus forms, bud formation can be encouraged by the use of the appropriate plant hormones according to known methods, and the buds are transferred to rooting medium for regeneration of plants.

The plants can then be used to establish 30 repetitive, or seed, generations or using vegetative propagation techniques.

Transgenic plants can also be generated without using tissue cultures.

Also considered part of this invention are transgenic plants, tree cells, yeast, bacteria, fungi, insects and animals containing the construction of a chimeric gene comprising a nucleic acid encoding recombinant HAO for the production of VLP, according to the presence - 5 te invention.

The regulatory elements of the present invention can also be combined with a coding region of interest for expression within a range of host organisms that are responsible for transformation, or transient expression.

Such organisms include, but are not limited to, plants, both monocots and dicots, for example, but not limited to, corn, cereal plants, wheat, barley, oats, Nicotiana spp, Brasslca spp, soybean, ugly, peas, alfalfa, potatoes, tomatoes, ginseng, and A-

- rabidopsis.

Methods for stable transformation and regeneration of these organisms are established in the art and known to one skilled in the art.

The method of obtaining transformed and regenerated pIantas is not critical to the present invention.

By "transformation" is significant the stable specific transfer of genetic information (nucleotide sequence) that is manifested genotypically, phenotypically, or both.

The interspecific transfer of genetic information from a chimeric construct to a host can be inherited, and the transfer of genetic information considered stable, or the transfer can be transient, and the transfer of genetic information is not hereditary. 25 By the term "plant material", any material derived from a plant is significant.

The plant material can comprise a total plant, tissue, cells, or any fraction thereof.

In addition, plant matter may comprise intracellular plant components, extracellular plant components, liquid or solid plant extracts, or a combination thereof.

In addition, the pianta material may comprise plants, plant cells, tissue, a liquid extract, or a combination thereof, from plant leaves, stems, fruit, roots, or a combination thereof. The plant material may comprise a plant or portion thereof that has not been individualized for any processing steps. However, it is also contemplated that the pIanta material can be individualized for minimum processing steps 5 as defined below, or more stringent processing, including purification of partial or substantial depletion using techniques commonly known within the art including, but not limited to, limited to, chromatography, electrophoresis and the like. By the term "minimal processing" is significant plant matter, for example, a plant or portion thereof comprising a protein of interest that is partially purified to produce a plant extract, homogenate, plant fraction homogenate, or the like (i.e. is minimally processed). Partial purification can understand, but

A is not limited to, disruption of plant cell structures, thereby creating a composition comprising soluble plant components, and insoluble plant components that can be separated, for example, but not limited to, by centrifugation, filtration, or a combination thereof. In this regard, proteins secreted within the extracellular space of leaf or other tissues can be readily obtained using vacuum or centrifugal extraction, or tissues can be extracted under pressure by passing through rollers or grinding or the like to press or release the free protein from inside the extracellular space. Minimal processing may also involve the preparation of crude extracts of soluble proteins, since these preparations would have reasonable contamination of secondary plant products. In addition, minimal processing involves aqueous extraction of a soluble protein from 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. 30 The plant material, in the form of plant material or tissue, can be orally distributed to an individual. Plant matter can be administered as part of a dietary supplement, along with other foods

or encapsulated.

The plant or tissue material can also be concentrated to improve or increase palatability, or provided with other materials, ingredients, or pharmaceutical excipients, as required. Examples of a target individual or organism that the VLPS of the present invention can be administered include, but are not limited to, humans, primates, birds, water birds, migratory birds, quail, duck, geese, animals domestic, hen, sulno, ewe, horse, camel, canine, dogs, feline, cats, tiger, leopard, cat 10, marten, stone marten, ferrets, domestic animals, farm animals, rabbits, mice, rats, guinea pigs, or other rodents, seals, whales, and the like.

Such target organisms are exemplary, not

- are to be considered as limiting the applications and uses of the present invention. 15 It is contemplated that a plant comprising the protein of

- interest, or that expresses the VLP comprising the protein of interest, can be administered to a target individual or organism, in a variety of ways, depending on the need and the situation.

For example, the protein of interest obtained from the plant can be extracted before use 20 in the form or raw, partially purified, or purified.

If the protein is to be purified, then it can be produced in any edible or non-edible plants.

In addition, if the protein is orally administered, the plant tissue can be collected and fed directly to the individual, or the collected tissue can be dried before feeding, or an animal can be allowed to graze on the plant with no previous collection taking place.

It is also considered within the scope of this invention the plant tissues collected to be provided as a food supplement within the animal's diet.

If the plant tissue is being fed to an animal with little or no further processing, it is preferred that the plant tissue being administered is edible.

Post-transcriptional gene silencing (PTGS) can be involved in the expression of transgenic limitation in plants, and coexpression of a silencing suppressor from potato Y virus (HcPro) can be used to counteract specific degradation of transgene mRNAs (Brigneti et al., 1998). Alternating silencing suppressors are well known in the art and can be used as described here (Chiba et 5 al., 2006, Virology 346: 7-14; which is incorporated by reference), for example, but not limited to , TEV -pVHC-Pro (Tobacco etch virus-pl / HC-Pro), BYV -p21, pl9 of tomato closed evolution virus (TBSV pl9), tomato wrinkle virus capsid protein (TCV -CP), 2b cucumber mosaic virus; CMV-2b), potato X virus p25 (PVX-p25), potato M virus p11 (PVM-pl 1), potato S virus pi 1 (PVS-pl 1), p16 virus blueberry species, (BSCV-pl6), p23 of Citrus tristexa virus (CTV-p23), p24 of virus-2 associated with vine leaf, (GLRaV-2 p24), plO of vine virus A, (GVA- plO), grape vine B virus p14 (GVB-p14), Heracleum latent virus plO (HLV-pIO), or Garlic common latent virus pl6 (GCLV-. 15 pl6). Therefore, a suppressor is silenced, for example, but not limited to

- do a, HcPro, TEV-pVHC-Pro, BYV-p21, TBSV pl9, TCV-CP, CMV-2b, PVX-p25, PVM-pll, PVS-pl 1, BScV-pl6, CTV-p23, GLRaV- 2 p24, GBV-p14, HLV-plO, GCLV-pl6 or GVA-plO, can be coexpressed together with the nucleic acid sequence encoding the protein of interest to ensure additionally high levels of protein production within a plant.

In addition, VLPS can be produced that comprise a combination of HA subtypes.

For example, VLPs can comprise one or more HA from the subtype Hl, H2, H3, H4, H5, H6, H7, H8, H9, HlO, Hll, H12, H13, H14, H15, H16, or a combination of them.

The selection of the SAH combination can be determined by the intended use of the vaccine prepared from the VLP.

For example, a vaccine for use in inoculating birds can comprise any combination of HA subtypes, while VLPs useful for inoculating humans can comprise subtypes of one or more than one of the H1, H2, H3, H5 subtypes. However, other HA subtype combinations can be prepared depending on the use of the VLP.

In order to produce VLPS comprising combinations of HA subtypes, the desired HA subtype can be coexpressed within the same cell, for example, a plant cell.

In addition, VLPS produced as described here do not include neuraminidase (NA). However, NA can be coexpressed with HA with VLPS comprising HA and NA can be desired. Therefore, the present invention additionally includes a suitable vector comprising the chimeric construct suitable for use with stable or transient expression systems.

Genetic information can also be provided within one or more than one construction.

For example, a nucleotide sequence that encodes a protein of interest can be introduced into a construct, and a second nucleotide sequence that encodes a protein that modifies glycosylation of the protein of interest can be introduced using a separate building.

These nucleotide sequences can then be coexpressed within a plant.

However, a construct comprising a 15 nucleotide sequence that encodes both the protein of interest and the protein that "modifies the glycosylation profile of the protein of interest can also be used.

In this case the nucleotide sequence would comprise a first sequence comprising a first nucleic acid sequence that encodes the protein of interest operably linked to a promoter or regulatory region, and a second sequence comprises a second nucleic acid sequence that encodes the protein that modifies the glycosylation profile of the protein of interest, the second sequence operably linked to a promoter or regulatory region.

By "coexpression" it is significant that two or more than two 25 nucleotide sequences are expressed at about the same time inside the plant, and within the same plant tissue.

However, nucleotide sequences do not need to be expressed at exactly the same time.

Preferably, the two or more nucleotide sequences are expressed in such a way that the encoded products have a chance of interacting.

For example, the protein that modifies glycosylation of the protein of interest can be expressed before or during the period when the protein of interest is expressed so that modification of the glycosylation of the protein

in the case of interest occurs.

Two or more than two nucleotide sequences can be coexpressed using a transient expression system, where the two or more sequences are introduced into the plant at about the same time under conditions that both sequences are expressed.

Alternatively, a platform plant comprising one of the nucleotide sequences, for example, the sequence encoding the protein that modifies the glycosylation profile of the protein of interest, can be transformed, either transiently, or into a stable manner, with an additional sequence encoding the protein of interest.

In this case, the sequence that encodes the protein that modifies the glycosylation profile of the protein of interest can be expressed within the desired tissue, during a desired stage of development, or its expression can be induced using a promoter. inducible, and the additional sequence encoding the. interest can be expressed under similar conditions and in the same tissue, 15 to ensure that the nucleotide sequences are coexpressed.

The constructs of the present invention can be introduced

, in plant cells using Ti plasmldeos, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques, see, for example, Weissbach and Weiss-20 bach, Methods for P / ant Mo / ecu / ar Bio / ogy, Academy Press, New York Vlll, pp. 421-463 (1988); Geierson and Corey, P / ant Mo / ecu / ar Bio / ogy, 2nd Ed. (1988); and Miki and lyer, Fundamentals of Gene Transfer in Plants. ln for Antibody / ism, 2d Ed.

DT.

Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Adonis Wesly, Langmans Ltd.

London, pp. 561-579 (1997). Other methods 25 include understanding direct DNA, the use of liposomes, electroporation, for example, using protoplasts, microinjection, microprojectiles or whiskers, and vacuum infiltration.

See, for example, Bilang, et al. (Gene 100: 247-250 (1991), Scheid et al. (Mol.

Gen.

Genet. 228: 104-112, 1991), Guerche et al. (PIanta Science 52: 111-116, 1987), Neuhause et al. (Theor.

Appl 30 Genet. 75: 30-36, 1987), Klein et al., Nature 327: 70-73 (1987); Howell et al. (Science 208: 1265, 1980), Horsch et al. (Science 227: 1229-1231, 1985), De8lock et al., Plant Physiology 91: 694-701, 1989), Methods for Plant Mole-

Biology (Weissbach and Weissbach, eds., Academic Press lnc., 1988), Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press lnc., 1989), Liu and Lomonossoff (J.

Virol Meth, 105: 343-348, 2002,), United States Patent Nos. 4,945,050; 5,036,006; and 5,100,792, United States Patent Application No. Serial No. 08 / 438,666, filed on May 10, 1995, and 07 / 951,715, filed on September 25, 1992, (all of which are incorporated herein by reference). Transient expression methods can be used to express the constructs of the present invention (see, Liu and Lomonossoff, 10 2002, Journal of Virological Methods, 105: 343-348; which is incorporated herein by reference). Alternatively, a vacuum-based transient expression method, as described by Kapila et al. 1997 (incorporated herein by reference) may be used.

These methods may include, for example, but are not limited to, an Agroinoculation or Agroinfiltration method,. 15 however, other transient methods can also be used as noted above.

With or Agroinoculation or Agroinfiltration, a mixture of

. Agrobacter / a comprising the desired nucleic acid enters the intercellular spaces of a tissue, for example, the leaves, aerial portion of the plant (including stem, leaves and flower), another portion of the plant (stem, root, flower), or 20 total plant.

After crossing the epidermis, Agrobacter / um infects and transfers copies of t-DNA in the cells.

The t-DNA is episomally transcribed and the mRNA translated, leading to the production of the protein of interest in infected cells; however, the passage of t-DNA into the nucleus is transient. If the nucleotide sequence of interest encodes a product that is directly or indirectly toxic to the plant, then by using the method of the present invention, such toxicity can be reduced throughout the entire plant by selectively expressing the sequence of interest of cleotide within a desired tissue or at a desired stage of plant development.

In addition, the limited period of expression resulting from transient expression can reduce the effect when a toxic product is produced in the plant.

An inducible promoter, a tissue specific promoter, or a cell specific promoter, can be used to selectively target expression of the sequence of interest. The recombinant HA VLPS of the present invention can be used in conjunction with existing influenza vaccines, to supplement vaccines, make them more effective, and to reduce the required dosages for administration. As would be known to one skilled in the art, the vaccine can be targeted against one or more than one flu virus. Examples of suitable vaccines include, but are not limited to, those commercially available from Sanofi-Pasteur, ID Biomedical, Merial, 10 Sinovac, Chiron, Roche, Medlmmune, GlaxoSmithKline, Novartis, Sanofi- Aventis, Serono, Shire Pharmaceuticals, and similar. If desired, the VLPs of the present invention can be mixed with a suitable adjuvant as one skilled in the art would be known. In addition, VLP can be used in a vaccine composition 15 comprising an effective dose of VLP for the treatment of an organ. target body as described above. In addition, the VLP produced according to. with the present invention can be combined with VLPS obtained using different flu proteins, for example, neuraminidase (NA). Therefore, the present invention provides a method for inducing immunity to influenza virus infection in a target animal or organism comprising administering an effective dose of a vaccine comprising one or more than one VLP. The vaccine can be administered orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. The administration of 25 VLPS produced in accordance with the present invention is described in Example

6. The administration of a plant-produced H5 VLP results in a significantly higher response when compared to the administration of soluble HA (see Figures 21A and 21B). As shown in Figures 26A and 26B, an individual administered with A / Indonesia / 5/05 H5 VLPs provides cross-protection to a challenge with influenza A / Peru / 582/06 (H5N1; "Peru H5N1"). The administration of Indonesia H5 VLPs prior to the challenge does not result in any loss of mas-

body.

However, in an individual not administered with H5VLPS, but challenged with Turkey H5N1, it exhibits significant loss of body mass, and several individuals die.

These data, therefore, demonstrate that VLPS of influenza produced by 5 of the plant comprising the H5 viral protein hemagglutinin induces a specific immune response to pathogenic influenza strains, and that virus-like particles can originate from a plant plasma membrane.

Therefore, the present invention provides a composition 10 comprising an effective dose of a VLP comprising an influenza virus HA protein, one or more than a plant lipid, and a pharmaceutically acceptable carrier.

The HA protein from influenza viruses

- could be H5 Indonesia / 5/2006. A method of inducing immunity to an influenza virus infection in an individual is also provided.

The method comprising administering the virus-like particle comprising an influenza virus HA protein, one or more plant liplde, and a pharmaceutically acceptable carrier.

The virus-like particle can be administered to an individual orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or 20 subcutaneously.

Compositions according to various embodiments of the invention may comprise VLPS of two or more strains of influenza or subtypes. "Two or more" refers to two, three, four, five, six, seven, eight, nine, 10 or more strains or subtypes.

The strains or subtypes represented can be 25 of a single subtype (for example, all H1N1, or all H5N1), or can be a combination of subtypes.

Exemplary subtypes and strains include, but are not limited to, those described herein (for example, A / New Caledonia / 20/99 (H1N1) A / Indonesia / 5/2006 (H5N1), A / chicken / New York / 1995, A / mallard / DE / 677/88 (H2 N8), A / Texas / 32/2003, A / mallard of the 30 northern hemisphere / MN / 33/00, A / duck / Shanghai / 1/2000, A / northern scribble / TX / 828189/02, A / Peru / Ontario / 6118/68 (H8N4), A / shoveler / frog / G54 / 03, A / chicken / Germany / N / 1949 (H10N7), A / duck / england / 56 (H11 N6),

A / duck / Alberta / 60/76 (H12N5), A / Wild duck / Maryland / 704/77 (H13N6), N Northern hemisphere wild duck / Gurjev / 263/82, A / duck / Australia / 341/83 (H15N8 ), Black-headed wildcat / Sweden / 5/99 (H16N3), B / Lee / 40, C / Johannesburg / 66, A / Puerto Rico / 8/34 (H1N1), N8risbane / 59/2007 (H1N1), 5 A / llhas Salomão 3/2006 (H1N1), A / 8risbane 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2), B / Malaysia / 2506/2004, B / Florida / 4/2006, NSingapura / 1/57 (H2N2), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / Hong-kong / W312 / 97 (H6N1), A / Equine / Prague / 56 (H7N7), A / Hong-kong / 1073/99 (H9N2)). 10 The choice of combination of strains and subtypes may depend on the geographical area of the individuals likely to be exposed to influenza, proximity of animal species to a human population to be

- immunized (for example, water bird species, agricultural animals such as swine, etc.) and the strains they carry, are exposed to or are>

15 likely to be exposed to prognoses of antigenic subtypes. within subtypes or strains, or combinations of these factors.

Examples of

. combinations used in the past years are available (see URL: who.int/csr/doença/gripe/vacina recommendations l / en). Some or all of these strains can be used in the 20 combinations shown, or in other combinations, in the production of a vaccine composition.

More particularly, exemplary combinations may include VLPS of two or more strains or subtypes selected from the group comprising: A / 8risbane / 59/2007 (H1N1), a virus similar to 25 A / 8risbane / 59/2007 (H1N1), A / Brisbane / 10/2007 (H3N2), a virus similar to A / 8risbane / 10/2007 (H3N2), B / Florida / 4/2006 or a virus similar to B / Florida / 4/2006. Another exemplary combination may include VLPS of two or more strains or subtypes selected from the group comprising 30 A / Indonesia / 5/2005, a virus similar to Indonesia / 5/2005, A / Vietnam / 1194/2004, a virus similar to A / Vietnam / 1194/2004, A / Anhui / 1/05, a virus similar to A / Anhui / l / 05, A / goose / Guiyang / 337/2006, virus similar to

Nganso / Guiyang / 337/2006, A / chicken / Shanxi / 2/2006, or a virus similar to Ngalinha / Shanxi / 2/2006. Another exemplary combination may include A / Chicken / ltaly / 13474/99 (H7 type) VLPs or A / Chicken / 8ritish Columbia / 04 (H7N3) flu strains. 5 Another exemplary combination may include NL Chicken / Hong-kong / G9 / 97 or NHong-kong / 1073/99 VLPS. Another exemplary combination may comprise VLPS by Ajllhas Salomão / 3/2006. Another exemplary combination may comprise VLPs from A / 8risbane / 10/2007. Another exemplary combination may comprise VLPS from A / Wisconsin / 67/2005. 10 Another exemplary combination may comprise VLPS of strains or subtypes 8 / Malaysia / 2506/2004, B / Florida / 4/2006 or B / 8risbane / 3/2007. The two or more VLPs can be expressed individually, and

- the purified or semi-purified VLPS subsequently combined.

Alternatively, VLPS can be coexpressed in the same host, for example, a plant.

VLPS can be combined or produced in a desired proportion, for example, about equivalent proportions,

. or they can be combined in such a way that a subtype or strain comprises most of the VLPS in the composition.

Therefore, the invention provides compositions comprising 20 VLPS of two or more strains or subtypes.

VLPS of enveloped viruses usually acquire their envelope from the membrane they originate from.

Plant plasma membranes have a phytosterol complement that can have immunostimulatory effects.

To investigate this possibility, VLPS H5 produced from plant were administered to animals in the presence or absence of an adjuvant, and HAI (hemagglutination inhibition antibody response) determined (Figures 22A, 22B). In the absence of H5 VLPs produced from pIanta added with adjuvant, it demonstrates significant HAI, indicative of a systemic immune response to antigen administration.

In addition, the profiles of the VLPS antibody isotype administered in the presence or absence of adjuvant are similar (Figure 23 A). Table 5 lists sequences provided in various embodiments of the invention.

Table 5: Sequence description for sequence identifiers.

SEQ ID No Description of Sequence In Develop 1 N-Terminal Hl fragment I Figure 5 to 2 C-Terminal Hl fragment i Figure 5b 3 H5 coding sequence I Figure 6 4 Plato-443c primer Figure 7a 5 SpHA (lnd) -Plast primer .r Figure 7b Plasto-SpHA (lnd) primer .c.

Figure 7c 7 HA primer (lnd) -Sac.r Figure 7d 8 Expression cassette sequence -., .— Figure 1 based on plastocyanine alfalfa used for the expression of Hl 9 HAI peptide sequence (A / New Figure 8a Caledonia / 20/99) 10 HA5 peptide sequence Figure 8b (A / Indonesia / 5/2006) 11 Influenza A Subtype H7 co-sequence Figure 9d (A / chicken / New York / l 995) 12 Influenza A Subtype H2 Co-sequence Figure 10O dification (A / wild duck / DE / 677/88 (H2N8)) 13 Influenza A Subtype H3 co-sequence Figure 12O dification (A / Texas / 32/2003) 14 Influenza A Subtype H4 co-sequence Figure 10Ocification (Northern hemisphere wildcat / MN / 33/00) 15 Influenza A Subtype H5 co-sequence figure (A / duck / Shanghai / 1/2000) 16 Influenza A Subtype H6 co-sequence Figure 10Odification (Northrabijunco do norte / TX / 828189/02) 17 Influenza A Subtype H8 co-sequence Figure 1Ofification (A / Peru / Ontario / 6118/68 (H8N4)) 18 Influenza Subtype H9 co-Figure 10O dification sequence (A / shoveler / frog / G54 / 03)

SEQIDNO I Description of Sequence at Revelation 19 I Influenza A Subtype H1O Figure sequence 10h coding | (A / ga | inha / A | germany / N / 1949 (HiON 7)) 20 I Influenza A Subtype Hll co-Figure 10i dification (A / duck / england / 56 (H11N6)) 21 jgripe a Subtype Hl2 co-sequence Figure (A / duck / Alberta / 60/76 (H 12N5)) 22 I Influenza A Subtype Hl3 co-sequence Figure 10 Okification (NPato / Maryland / 704/77 (H13N6)) 23 i Influenza A Subtype H14 sequence of Figure 101 I coding (NPhilateral Northern Hemisphere / Gurjev / 263/82) 24 I Influenza A Subtype Hl5 sequence Figure 1O lification (A / duck / Australia / 341/83 (h15n8)) 25 I Influenza A Subtype H16 H16N3)) 26 I Flu B HA coding sequence Figure 10o (8 / Lee / 40) 27 I Flu C HA coding sequence Figure 10O (C / Johannesburg / 66) 28 i Complete HAO Hl sequence Figure 5c 29 I lniciator Xmal- pPlas.c Figure lOq 30 I lniciator Sacl-ATG-pplas.r Figure lOr 31 lniciator Sacl-PlasTer.c Figure lOs 32 ecoRI-PlasTer.r Figure lOt 33 I A / New Caledonia / 20/99 (H1N1) Figure 16 Gen8ank Accession No.

AY289929 34 M.

Sativa protein disulfide isomerase Figure 17 Gen8ank Accession No.

Z11499 35 I N.Porto Rico / 8/34 (H1N1) Gen8ank Figure 18 Accession No.

NC _002016.1

SEQIDNO I Description of Sequence I In Revelation 36 I CIone 774: DNA from Dralll to Sacl with- I Figure 28 comprising regulatory region of) plastocyanin operatively linked to I isequence encoding HA of 59 / 2007j (H1N1) 37 i Clone 775: DNA from Dralll to Sacl with- I Figure 29 comprising regulatory region of I plastocyanin operatively linked to II sequence encoding HA of Nllhas I Salomão 3/2006 (H1N1) 38 | Clone 776: DNA from Dralll to Sacl with- I Figure 30 comprising regulatory region of) plastocyanin operatively linked to I HA-coding sequence from A / 8risbane 10/2007 (H3N2) 39 I Clone 777: DNA from Dralll to Sacl with- Figure 31 comprising regulatory region of) plastocyanin operatively linked to I HA-encoding sequence of A / Wisconsin / 67/2005 (H3N2) 40 I Clone 778: DNA from Dralll to Sacl with Figure I comprising regulatory region of) operatively linked plastocyanin the I HA-coding sequence from B / Malaysia / 2506/2004 41 Clone 779: DNA from Dralll to Sacl with- Figure 33 comprising the regulatory region of I plastocyanin operatively linked to the HA-coding sequence from B / Florida / 4/2006 42 Clone 780: DNA from Dralll to Sacl with- Figure 34 comprising regulatory region of plastocyanin operatively linked to I sequence that encodes HA from A / Singapore / 1/57 (H2N2)

SEQIDNO I Description of Sequence In Revelation 43 I Clone 781: DNA from Dralll to Sacl- Figure 35 comprising regulatory region of plastocyanin operatively linked to the HA coding sequence of A / Anhui / 1/2005 (H5N1) 44 | Clone 782: Dralll aSacl DNA with- Figure 36 comprising regulatory region of I plastocyanin operatively linked to the HA coding sequence of A / Vietnam / 1194/2004 (H5N1) 45 I Clone 783: Dralll DNA to Sacl with- Figure 37 regulatory region of) plastocyanin operably linked to the HA-coding sequence of A Cer- »

ceta / Hong-kong / W312 / 97 (H6N1) 46 I CIone 784: DNA from Dralll to Sacl with- Figure 38 comprising regulatory region of plastocyanin operatively linked to the HA / Equine / Prague / 56 (H7N7) coding sequence 47 i Clone 785: DNA from Dralll to Sacl with- Figure 39 comprising regulatory region of I plastocyanin operatively linked to the A / Hong-kong / 107 3/99 (H9N2) HA coding sequence 48 I Clone 74 Amino acid sequence Figure 40A I from HA A / 8risbane / 59/2007 (H1N1) 49 I Clone 775 HA amino acid sequence Figure 40B! Nllhas Salomão 3/2006 (H1N1) acid 50 I Clone 776 HA amino acid sequence Figure 41A N8risbane acid 10/2007 (H3N2) 51 I Clone 777 HA amino acid sequence Figure 4 IB acid A / Wisconsin / 67/2005 ( H3N2) 52 I Clone 778 HA amino acid sequence- Figure 42A acid B / Malaysia / 2506/2004 53 Clone 779 HA amino acid sequence- Figure 42B acid B / Florida / 4/2006

SEQIDNO I Description of Sequence In Develop 54 l Clone 780 HA amino acid sequence- Figure 43A acid / Singapore / 1/57 (H2N2) 55 I Clone 781 HA amino acid sequence- Figure 43 B acid A / Anhui / 1/2005 (H5N1 ) 56 i Clone 782 HA amino acid sequence Figure 44A acid A / Vietnam / 1194/2004 (H5N1) 57 | CIone 783 HA amino acid sequence Figure 44B A / Teal / Hong-kong / W312 / 97 (H6N1) 58 l Clone 784 HA amino acid sequence- Figure 45A A / Equine / Prague / 56 (H7N7) 59: Clone 785 HA amino acid sequence Figure 45B acid A / Hong-kong / 1073/99 (H9N2) 60 I HA expression cassette comprising Figure 51 l plastocyanin alfalfa promoter and 5 'UTR, hemagglutinin coding sequence H5 AI / Indonesia / 5/2005 (Construction # | 660), plastocyanine alfalfa 3 'RTU and terminator sequences —'-- · --- 61 I HA expression cassette comprising Figure 52 giving promoter for plastocyani- alfalfa | na and 5 'UTR, hemagglutinin I / A coding sequence from A / Nova Caldonia / 20/1999 (Construction # 540), I alfalfa plastocyanin 3' UTR and terminating sequences 62 HA expression cassette comprising Figure 53 l giving promoter of plastocyanin alfalfa and 5 'UTR, H8 hebglutinin coding sequence from N8risbane / 59/2007 (construction # 774), plastocyanin alfalfa 3' UTR and terminating sequences

SEQIDNO I Description of Sequence I In Revelation 63 l HA expression cassette comprising: Figure 54 l giving promoter of plastocyanin alfalfa and 5 'UTR, coding sequence of H I of Nllhas II Solomão / 3/2006 (Hl Nl) (construction) l # 775), 3 'UTR plastocyanin alfalfa and terminator sequences 64 HA expression cassette comprising Figure 55 I plastocyanin alfalfa promoter and 5' UTR, hemagglutinin coding sequence for H2 from i A / Singapore / 1/57 (H2N2) (construction II # 780), plastocyanine alfalfa 3 'RTU and I terminator sequences 65 HA expression cassette comprising Figure 56 I promoting plastocyanine alfalfa and I | na and 5 'UTR, IA / Anhui / 1/2005 (H5N1) H5 hemagglutinin coding sequence (Construction * i I 781), plastocyanin alfalfa 3' RTU and I terminating sequences - · '66 l HA expression cassette comprising I Figure 57 I giving plastocyanin alfalfa promoter and 5 'UTR, H5 hemagglutinin coding sequence de) A / Viet Nam / 1194/2004 (H5N1) (Construction # 782), 3 'RTF plastocyanin alfalfa and terminator sequences 67 HA expression cassette comprising Figure 58 i plastocyanid alfalfa promoter and 5' RTU, A / Teal / Hong Kong / W312 / 97 I (H6N1) H6 hemagglutinin coding sequence (Construction # 783), 3 'RTU plastocyanin alfalfa and terminating I sequences

SEQIDNo: Description of Sequence I in Revelation 68 | HA expression cassette comprising I Figure 59: plastocyanin alfalfa promoter and 5 'UTR, coding sequence | H / H9 hemagglutinin I from A / Hong II Kong / 1073/99 (H9N2) (Construction I) # 785), 3 'RTU plastocyanin alfalfa and 69 HA expression cassette terminator comprising Figure 60) giving plastocyani alfalfa promoter - I | na and 5 'UTR, H3 hemagglutinin coding sequence of 1A / 8risbane / 10/2007 (H3N2), alfalfa | I 3 'UTR plastocyanin and I terminator sequences 70 HA expression cassette comprising Figure 61 I plastocyanin alfalfa promoter and 5' UTR, IA / Wisconsin / 67/2005 H3 hemagglutinin encoding sequence (H3N2 ), alfalfa | I 3 'UTR plastocyanin and I terminator sequences 71 HA expression cassette comprising Figure 62 I plastocyanin alfalfa promoter and 5' UTR, NEquino / Praga / 56 (H7N7) H7 hemagglutinin coding sequence, alfalfa l 3 'RTU plastocyanin and I terminator sequences 72 HA expression cassette comprising Figure 63 - I promoter of plastocyanin-I alfalfa prognosis | na and 5' UTR, HA hemagglutinin coding sequence from IB / Malaysia / 250672004, alfalfa pIastoci- i janina 3 'RTU and final sequences

SEQIDNO) String Description | In Revelation 73 i HA expression cassette comprising I Figure 64 I promoting a plastocyanin alfalfa promoter and 5 'UTR, HA hemagglutinin coding sequence from I B / FI / 4/2006, plastocyanine alfalfa | I 3 'RTU and terminating sequences | 74 j seq id no: 49.48, 33 e | Figure 65 9 75 Hl New amino acid sequence Figure 67 I Caledonia (AAP34324.1) encoded I by SEQ ID NO: 33 76 Hl Porto amino acid sequence Figure 68: Rico (NC _ 0409878.1) encoded by i SEQ ID NO: 35 The invention will now be described in detail with reference to the following non-limiting examples only. Methods and Materials

1. Set of expression cassettes "5 All manipulations were done using the general molecular biology protocols of Sambrook and Russell (2001; which is incorporated by reference here). The first cloning step consists of the assembly of a receptor plasmid containing regulatory elements upstream and downstream of the alfalfa pIastocyanin gene The plastocyanin promoter and 10 5'UTR sequences were amplified from alfalfa genomic DNA using Xmal-pPlas.c oligonucleotide primers (SEQ ID NO: 29; Figure IOQ) and Sacl-ATG-pPlas.r (SEQ ID NO: 30; Figure IOR). The resulting amplification product was digested with Xmal and Sacl and ligated into pCAMBIA2300 (Cambia, Canberra, Australia), previously digested with the same enzymes, to 15 create pCAMBIApromo PIasto. Similarly, the 3'UTR plastocyanin gene sequences and terminator were amplified from alfalfa genomic DNA using the following primers: Sacl-Plas7er.c (SEQ ID NO: 31; Figure lOS) and EcoRl- PlasTer.r (SEQ ID NO: 32; Figure IOT), and the product was digested with Sacl and EcoRI before being inserted into the same places as pCAMBI-20 ApromoPlasto to create pCAMBIAPlasto.

The open reading frame of the H1 influenza A / New Caledonia / 20/99 (Hl Nl) gene was synthesized into two fragments (Planta Biotechnology Institute, National Research Council, Saskatoon, Canada). A first synthesized fragment corresponds to the wild type H1 5 coding sequence (Gen8ank ace. No. AY289929; SEQ ID NO: 33; Figure 16) lacking the signal peptide coding sequence at the 5 'end and the coding sequence of transmembrane domain at the 3 'end. A Bglll restriction site was added at the 5 'end of the coding sequence and a dual Sacl / Stul site was added immediately downstream of the stop codon at the 3' end of the fragment to produce SEQ ID NO: 1 (Figure 5A). A second fragment encoding the C-terminal end of the H1 protein (comprising - a transmembrane domain and cytoplasmic tail) from the Kpnl site to the stop codon, and flanked 3 'by Sacl and 15 Stul restriction sites was also synthesized (SEQ ID NO. 2; Figure 5B). The first Hl fragment was digested with Bglll and Sacl and cloned at the same sites as a binary vector (pCAMBIAPlasto) containing the plastocyanin and 5'UTR promoter fused to the signal peptide! alfalfa gene protein disulfide isomerase (PDl) gene (nucleotides 32-103; Accession No. 20 Z11499; SEQ ID NO: 34; Figure 17) resulting in a chimeric PDl-Hl gene downstream of the plastocyanin regulatory elements. The sequence of the plastocyanine-based cassette containing the signal peptide PD1 is shown in Figure 1 (SEQ ID NO: 8). The resulting pIasmid contains the H1 coding region fused to the PD1 signal peptide and flanked by the plastocyanin regulatory elements. The addition of the C-terminal end coding region (which encodes the transmembrane domain and the cytoplasmic tail) was obtained by inserting the synthesized fragment (SEQ ID NO: 2; Figure 5B) previously digested with Kpnl and Sacl, in the plasmid of expression H1. The resulting plasmid, called 540, is shown in Figure 11 (also see Figure 2A).

2. H5 expression cassette set A fragment encoding hemagglutinin from the flu strain

A / Indonesia / 5/05 (H5N1; Ace.

At the.

LANLISDN125873) was synthesized by Epoch Biolabs (Sugar Land, TX, USA). The fragment produced, containing the complete H5 coding region including the native signal peptide flanked by a Hindlll site immediately upstream of the initial ATG, and 5 a Sacl site immediately downstream of the stop codon (TAA), is presented in SEQ ID NO: 3 (Figure 6). The H5 coding region was cloned into an expression cassette based on pIastocyanin by the PCR-based ligation method presented in Darveau et al. (1995). Briefly, a first PCR amplification was obtained using PIatoLO 443C (SEQ ID NO: 4; Figure 7A) and SpHA (lnd) -Plasto.r (SEQ ID NO: 5; Figure 7B) and pCAMBIA promoPlasto primers as feedback.

In parallel, a second amplification was performed with Plasto-SpHA (lnd) .c primers (SEQ ID NO:

- 6; Figure 7C) and HA (lnd) -Sac.r (SEQ ID NO: 7; Figure 7D) with H5 coding fragment as a template.

The amplification obtained from both reactions 15 were mixed and the mixture served as a template for a third reaction (pooling reaction) using Plato-443c (SEQ ID NO: 4; Figure 7A) and HA (lnd) -Sac.r (SEQ ID NO: 7; Figure 7D) as primers.

The re- fragment. The result was digested with BamHl (in the plastocyanin promoter) and Sacl (at the 3rd end of the fragment) and cloned in pCAMBIAPlasto previously digested with the same enzymes.

The resulting plasmid, called 660, is shown in Figure 2B (see also Figure 11). The cassette encoding the soluble form of Hl was prepared by replacing the region encoding the transmembrane domain and the cytoplasmic tail # 540 with a fragment encoding the zip-25 leucine zipper per GCN4 p11 variant (Harbury et al., 1993 , Science 1993; 262: 1401- 1407). This fragment was synthesized by flanking Kpnl and Sacl sites to facilitate cloning.

The plasmid resulting from this substitution was named 544 and the expression cassette is illustrated in figure 11. A fusion between the tobacco etch virus (TEV) 5'UTR and the open reading structure of the Flu A / PR / gene 8/34 MI (Ace. # NC _002016) was synthesized with a Sacl site flanking added downstream from the stop codon.

The fragment was digested with Swal (in TEV 5'UTR) and Sacl,

and cloned into an expression cassette based on 2X35S / TEV in a binary plasmid pCAMBIA. The resulting plasmid perforates the M1 coding region under the control of a 2X35S / TEV and 5'UTR promoter and the NOS terminator (construction 750; figure 11). 5 An HcPro construction (35HcPro) was prepared as described in Hamilton et al. (2002). All clones were sequenced to confirm the integrity of the constructs. Plasmids were used to transform Agrobacteium tumefaciens (AGLI; ATCC, Manassas, VA 20108, USA) by electroporation 10 (Mattanovich et al., 1989). The integrity of all A. tumefaciens strains was confirmed by restriction mapping.

3. Preparation of plant biomass, inoculum, filtration, and collection for tapirs of Nicotiana benthamiana or Nicotiana tabacum were grown from seeds on flat surfaces filled with a substrate of commercial peat moss. The plants were allowed to grow in the greenhouse under a 16/8 photoperiod and at a temperature regime of 25 ° C day / 20 ° C night. Three weeks after sowing, the individual plants were capped. transplanted in pots and allowed to grow in the greenhouse for three additional weeks under the same environmental conditions. Before transformation, apical and axillary seedlings were removed at various times as indicated below, either by tightening the seedlings from the plant, or by chemical treatment of the plant. Agrobacteria transfected with 660, 540, 544, 750 or 35SHcPro constructs were grown in a YEB medium supplemented with 10 mM 2-25 [N-morpholino] ethanesulfonic acid (MES), 20 µM acetosiringone, 50 µg / ml kanamycin and 25 µg / ml carbenicillin p H 5.6 until they reach an OD600 between 0.6 and 1.6. Agrobacterium suspensions were centrifuged before use and resuspended in infiltration medium (10 mM MgC | 2 and 10 mM MES pH 5.6). Syringe filtration was performed as described 30 by Liu and Lomonosossoff (2002, Journal of Viro / ogica / Methods, 105: 343-348). For vacuum infiltration, suspensions of A. tumefaciens were centrifuged, resuspended in the infiltration medium and stored overnight at

4 ° C. On the day of infiltration, the batches of culture were diluted in 2.5 volumes of culture and allowed to warm up before use. The total plants of N. benthamiana or N. tabacum were placed upside down in the bacterial suspension in an air-tight stainless steel tank under a vacuum of 2.67 kPa at 5.14 kPa (20-40 Torr ) for 2 min. Following the infiltration of a syringe or vacuum, the plants were returned to the greenhouse for 4 to 5 days of incubation period until collection.

4. Leaf sampling and total protein extraction Following the incubation, the aerial part of the plants was collected, 10 frozen at -80 ° C, crushed into pieces. The total soluble proteins were extracted by homogenization (Polytron) each sample of frozen crushed material of plant material in 3 cold 50 mM volumes of Tris pH 7.4. 0.15 M NaCl, and 1 mM phenylmethanesulfonyl fluoride. After homogenization, the slurries were centrifuged at 20,000 g for 20 min at 4'C 15 and these crude extracts cleared (supernatant) kept for analysis. The total protein content of crude extracts was determined by assay

Bradford V (81o-Rad, Hercules, CA) using bovine serum albumin as the reference standard.

5. Protein extract size exclusion chromatography 20 High resolution drops from 32 ml Sephacryl "S-500 size exclusion chromatography (SEC) columns (S-500 HR: GE Health-care, Uppsala, Sweden, Cat. No. 17-0613-10) were packed and equilibrated with equilibration / elution buffer (50 mM Tris pH8, 150 mM NaCl). One milliliter and medium of crude protein extract was loaded onto the column, followed by an elution step with 45 ml of equihbrio buffer / elution.The elution was collected in fractions of 1.5 ml of relative content of elongated fractions was monitored by mixing 10 µL of the fraction with 200 µL diluted 81o-Rad protein dye reagent (81o-Rad, Hercules, CA). The column was washed with 2 column volumes of 0.2 N NaOH, followed by 10 30 column volumes of 50 mM Tris pH8, 150 mM NaCl, 20% ethanol Each separation was followed by a column calibration with Blue Dextran 2000 (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA ). The Blue Dextran 2000 elution profiles and soluble host proteins were compared between each separation to ensure uniformity of the elution profiles between the columns used.

6. Protein Analysis and "lmunoblottinq" 5 Protein concentrations were determined by the BCA protein assay (Pierce Biochemicals, Rockport IL). The proteins were separated by SDS-PAGE under conditions of reduction and stained with Co-massie Blue. The stained gels were scanned and densitometry analysis performed using lmagej Software (NIH). 10 The elution fraction of SEC elution was precipitated with acetone (Bollag et al., 1996), resuspended in 1/5 volume in equilibrium / elution buffer and separated by SDS-PAGE under reduction conditions. and electrotransferred on polyvinylene difluoride (PVDF) membranes (Roche Diagnostics Corporation, Indianapolis, IN) for immunodetection. Before 15 immunoblotting, the membranes were blocked with 5 ° /, skimmed milk "and 0.1 ° / o of Tween-20 in Tris-buffered saline (TBS-T) for 16-18 V hours at 4 ° C "Immunobiotting" was performed by incubation with a suitable antibody (Table 6), in 2 µg / ml in 2% skimmed milk in TBS-Tween 20 0.1%. The secondary antibodies used for detecting chemiluminescence were as indicated in Table 4, diluted as indicated by 2 ° /, of skimmed milk in TBS-Tween 20 0.1 ° /). Immunoreactive complexes were detected by chemiluminescence using luminol as the substrate (Roche Diagnostics Corporation). human IgG antibody enzyme was performed using the EZ-Link Plus® Activated Peroxidase conjugation kit (Pierce, Rockford, IL).

Table 6: Electrophoresis conditions, antibodies, and dilutions for "immunoblotting" of expressed proteins.

HA Condition Strain Antibody Dilution Antibody Dilution Subtype electro-primary secondary forphoresis rio Hl A / 8risban Reduction Fll10-150 4 µg / ml Goat 1: 100 and / 59/2007 antica- (H1N1) mumdon- go (JIR 115 -035- 146) Hl Nllhas Reduction NIBSC 1: 2000 Rabbit 1: 100 Salo- 07/104 anti-hand / 3/20 sheep 06 (JIR 313- (H1N1) 035-045) Hl A / New reduction FII10-150 4 Ug / ml Goat 1: 100 Caledô- antica- nia / 20/99 mundon- '(H1N1) go (JIR 115-035- 146) H2 NSingap Non-NIBSC 1: 1000 Rabbit 1: 100 ura / 1/57 reduction 00 / 440 anti- (H2N2) sheep (JIR 313-035-045) H5 Nlndonés ITC Reduction IT- 1: 4000 Goat 1: 100 ia / 5/2005 003-005V anti- (H5N1) Rabbit (JIR111- 035-144) H5 A / Anhui / 1 Reduction NIBSC 1: 750 Rabbit 1: 100/2005 07/338 anti- (H5N1) sheep (JIR 313-035-045)

HA Condition Strain Antibody Dilution Antibody Dilution Subtype electro-primary flu secondary H5 A / Vietnam / Non-itc rr-003- 1: 2000 Goat 1: 100 1194/200 reduction 005 anti- 4 (H5N1) Rabbit ( JIR111- 035-144) H6 A / Teal Non- BEINR 1: 500 Rabbit 1: 100 / Hong 663 anti-Kong / W3 sheep reduction 12/97 (JIR 313- (H6N1) 035-045) H9 A / Hong NIBSC reduction 1: 1000 Rabbit 1:10 000 Kong / 107 07/146 anti- 3/99 sheep (H9N2) (JIR 313-035-045) F11: Fitzgerald International Industries, Concord, MA, USA; NISBIC: National Institute for Biological Standards and Control; jIR: Jackson ImmunoResearch, West Grove, PA, USA; BEINR: Biodefense and emerging infections research resources 5 repository; ITC: Immune Technology Corporation, Woodside, NY, USA; H5 hemagglutination assay was based on a method described by Nayak. and Reichl (2004). Briefly, serial serial dilutions of the test samples (100 µL) were made in 96-well V-bottom microtiter plates containing 100 µL of PBS, leaving 100 µL of diluted samples per well.

One hundred microliters of a 0.25% turkey red blood cell suspension (Bio Link lnc., Syracuse, NY) was added to each well, and the plates were incubated for 2 hours at room temperature.

The reciprocal of the highest dilution showing complete haemagglutination recorded as AH activity.

In parallel, a recombinant HA standard (A / Viet Nam / l 203/2004 H5N1) (Protein Science Corporation, Meriden, CT) was diluted in PBS and operated as a control on each plate.

7. Ultracentrifugation of sucrose qradient One milliliter of fractions 9, 10 and 11 eluted from gel filtration chromatography on biomass containing H5 were pooled, loaded in 5 a discontinued sucrose density gradient of 20-60% (p / v), and centrifuged 17.5 hours at 125,000 g (4 ° C). The gradient was fractionated into 19 3-ml fractions starting at the top, and dialyzed to remove sucrose before immunological analysis and hemagglutination assays. 10 8. Electron miscroscopy SEC-eluting fractions to be observed by electron microscopy (EM) were first concentrated using 30 MWCO of ultrafiltration units (Millipore, Billerica, MA, USA). The concentrated fractions were fixed in PBS pH 7.4 containing 2 ° /, of glutaraldehyde for 24 hours at 4 ° C. Once fixed, the samples were adsorbed on 200 mesh nickel gratings coated with Formvar (Canemco, Lakefield, Canada) for 2 min, and the gratings were washed twice with de- ionized water. before being stained in 1 ° /, of phosphotungstenic acid. Observation was carried out under electron microscopy transmission at magnifications ranging from 10 OOOX to 150.0OOX (for images in Figures 4A and 4B). Alternatively, one hundred microliters of samples to be examined were placed in an Airfuge centrifuge tube (Beckman instruments, Palo Alto, CA, USA). A grid was placed at the bottom of the tube which was then centrifuged for 5 min at 120,000 g. The grid was removed, gently dried, and placed in a 3 ° / 0 drop of phosphotungstenic acid at pH 6 for staining. The grids were examined using a Hitachi 7100 (TEM) transmission electron microscope (for images in Figures 14B, 15B and 15C). For images in Figure 19, Leaf blocks of approximately 30 t and 1 mm3 were fixed in PBS containing 2.5% glutaraldehyde and washed in PBS containing 3 ° /, of sucrose before a post-fixation step in 1.33 % osmium tetroxide. Fixed samples were embedded in resin

Spurr and ultra-thin layers were laid on a grid. The samples were positively stained with 5 ° / 0 uranyl acetate and 0.2% lead citrate before observation. The grids were examined using a Hitachi 7100 transmission electron microscope (TEM). 5 9. Plasma membrane lipid analysis Plasma membranes (PM) were obtained from tobacco leaves and BY2 cells cultured after cell fractionation according to Mon- grand et al. by dividing a two-phase system of aqueous polymer with polyethylene glycol 3350 / dextran T-500 (6.6% each). All steps 10 were performed at 4 ° C. the lipids were extralated and purified from different fractions according to Bligh and Dyer. Polar and neutral lipids were separated by monodimensional HP-TLC using the solvent systems described in Lefebvre et al. Lipoids from PM fractions were detected after staining with copper acetate as described by Macala et al. Lipids were identified by comparing their migration time with those

V of standards (all standards were obtained from Sigma-Aldrich, St-Louis, MO, USA, except for SG which was obtained from Matreya, Pleasant Gap, PA, U'SA).

10. Purification of H5 VLP 20 660 frozen infiltrated leaves of N. benthamiana were homogenized in 1.5 volumes of 50 mM Tris pH 8, 150 mM NaCl and 0.04% sodium meta bisulfide using a commercial mixer. The resulting extract was supplemented with 1 mM PMSF and adjusted to pH 6 with 1 M acetic acid before being heated to 42 ° C for 5 min. Diatomaceous earth 25 (DE) was added to the heat-treated extract to adsorb contaminants precipitated by changing pH and heat treatment, and the slurry was filtered through a Whatman paper filter. The resulting bleached extract was centrifuged at 10,000 xg for 10 minutes at room temperature to remove residual DE, passed through 20 0.8 / 0.2 filters in an Acro-30 pack and loaded into a fetuin-agarose affinity column (Sigma-Aldrich , St-Louis, MO, USA). Then the washing step in 400 mM NaCl, 25 mM Tris pH 6, bound proteins were eluted with 1.5 M NaCl,

50 mM MES pH 6. The eluted VLP was supplemented with Tween-80 at a final concentration of 0.0005% (v / v). The VLP was concentrated in a 100 kDa MWCO Amicon membrane, centrifuged at 10,000 xg for 30 minutes at 4 ° C and resuspended in PBS pH 7.4 with 0.01% Tween-80 and 0.01% of 5 thimerosal. The suspended VLPS were filtered-sterilized before use.

11. Animal studies Mouse Studies on the immune response to the administration of influenza VLP were carried out with female BALB / C mice 6-8 weeks old (Charles River Laboratories). Seventy mice were randomly divided into fourteen groups of five animals. All groups were used for intramuscular immunization and six groups were used to test intranasal route of administration. All groups were immunized in a two-dose regimen, impulse immunization being done 3 weeks after the first immunization. For intramuscular administration in doe legs, mice

Non-anesthetized dongs were immunized with the plant-produced VLP H5 vaccine (0.1, 1, 5 or 12 cut), or a control hemagglutinin antigen (HA). The control HA comprises recombinant soluble hemagglutinin 20 produced based on strain A / Indonesia / 5/05 H5N1 and purified from 293 cell cultures (mmune Technology Corp., New York, USA) (used at 5 µg per injection less otherwise indicated) - Buffer control was PBS. These antigen consists of amino acids 18-530 of the HA protein, and has a His-tag and a modified cleavage site. 25 electron microscopy confirmed that this commercial product is not in the form of VLPS. To measure the adjuvant effect, two groups of animals were immunized with 5 µg of VLP H5 vaccine produced per plant plus a 2% Alhydrogel volume (alum, Accurate Chemical & Scientific Corporation, Westbury, NY, US) or with 5 µg of purified recombinant hemagglutinin 30 out of 293 cell cultures plus 1 volume of alum. Seventy mice were randomly divided into fourteen groups of five animals. Eight groups were used for intramuscular immunization and six groups were used to test intranasal route of administration.

All groups were immunized according to a pulse regime, the pulse immunization performed 3 weeks after the first immunization.

For intramuscular administration in doe legs, 5 non-anesthetized mice were immunized with the plant-produced H5 VLP (0.1, 1, 5 or 12 µg), or the control hemagglutinin antigen (HA) (5 µg ) or PBS.

All antigen preparations were mixed with 1/0 Alhydrogel (alum, Accurate Chemical & Scientific Corporation, Westbury, NY, US) in a 1: 1 volume ratio prior to immunizations.

In order to measure the adjuvant effect, two groups of animals were immunized with either 5 VLP H5 vaccine produced from a cutting pIanta or with 5 µg of control HA antigen without any adjuvant.

For intranasal administration, mice were briefly anesthetized by inhaling isoflurane using an automatic induction chamber.

They were then immunized by adding 4 µl of goat "/ nostril with the plant-produced VLP vaccine (0.1 or 1 µg), or with control HA antigen (1 µg) or with PBS.

All antigen preparations were mixed with chitosan glutamate 1 ° / 0 (Protosan, Novamatrixl FMC BioPolymer, Norway) before immunizations.

The mice in 20 then breathed in the solutions.

To verify the adjuvant effect with the intranasal route of administration, two groups of animals were immunized with 1 µg of VLP H5 vaccine produced from plants or with 1 µg of control antigen HA.

Ferrets 25 Ten groups of 5 ferrets (male, 18-24 weeks old, mass of approximately 1 kg) were used.

The treatment for each group is as described in Table 7. The adjuvant used was Alhydrogel (alum) (Superfos Biosector, Denmark) 2% (final = 1%). The vaccine composition was VLPS from A / Indonesia / 5/05 (H5N1) associated with membrane produced as described.

The vaccine control (positive control) was a H5 recombinant bound to the glycosylated membrane of an Indonesian strain produced using adenovirus in 293 cell cultures by lmmune Technology

Corporation (ITC). Table 7. Treatment groups Group n Product injected at a- Admission route I Adjuvant animals administration 1 5 PBS (negative control) i.m. * 2 I 5 I Vaccine-plant, 1 µg i.m. ~ 3 I 5 i Vaccine-plant, 1 µg i.m.

Alum 4 i 5 i Vaccine-plant, 5 µg i.m. m

5 i 5 I Vaccine-plant, 5 µg— i.m.

Alum 6 I 5: Plant vaccine, 7.5 µg i.m. ~ 7 i 5 I Plant vaccine, 15 µg i.m. 8 I 5) Plant vaccine, 15 µg i.m.

Alum 9 I 5 i Vaccine-plant, 30 µg i.m. 10 I 5 I Vaccine control, 5 µg i.m. i.m .: intramuscular Ferrets were evaluated for total health and appearance (body weight "5, rectal temperature, posture, skin, movement models, breathing

. excrement) regularly during the study.

The animals were immunized by intramuscular injection (0.5-1.0 total volume) in quadriceps on days 0, 14 and 28; for protocols incorporating adjuvant, the vaccine composition was combined with Alhydrogel just prior to immunization in a 10: 1 volume ratio). Serum samples were obtained on day 0 before immunization, and on day 21 and 35. Animals were sacrificed (aorea / cardiac perforation) on days 40-45, and spleens were collected and necropsy performed.

Anti-influenza antibody titers can be quantified in 15 ELISA assays using homologous or inactivated heterologous H5N1 viruses.

Hemagglutination inhibitory antibody titers of serum samples (pre-immune, day 21 and day 35) were evaluated by microtiter HAI as described (Aymard et al 1973). Briefly, the sera were pretreated with receptor-killing enzyme, inactivated with 20 heat and mixed with a suspension of erythrocytes (RBC-washed red blood cells). Lampire horse washed RBC (10 ° / j) are re-

recommended and considering that the test may vary depending on the source of the RBC (horse-dependent), RBCS washed from 10 horses were tested to select the most sensitive batch.

Alternatively, turkey RBC can be used.

Antibody titration was expressed as reciprocal 5 of the highest dilution which completely inhibits hemagglutination.

Cross-reactive HAI titrations: HAI titrations of ferrets immunized with a vaccine for A / Indonesia / 5/05 (clade 2.1) were measured using inactivated H5N1 strains of influenza from another subclade or clade such as clade 1 Vietnam strains A / Vietnam / l 203/2004 and 10 A / Vietnam / 1194/2004 or A / Anhui / O1 / 2005 (subclade 2.3) or A / turkey / Turkey / 1/05 (subclade 2.2). All analyzes were performed on individual samples.

Data analysis: Statistical analysis (ANOVA) will be performed on all data to establish whether differences between groups are statistically significant.

Experimental design for lethal challenge (mice) One hundred and twenty-eight mice were randomly divided. out of sixteen groups of eight animals, one group being non-immunized then challenged (negative control). All groups were immunized via intramuscular administration in a two-dose regimen, the second immunization being performed two weeks after the first immunization.

For intramuscular administration in deer legs, non-anesthetized mice were immunized with H5 VLP produced from plant (1, 5 or 15 µg), or 15 µg of HA or PBS control antigen.

All 25 antigen preparations were mixed with a 1 ° / 0 volume of Alhydrogel prior to immunizations (alum, Accurate Chemical & Scientific Corporation, Westbury, NY, US). During the immunization period, the mice were weighed once in a week and observed and monitored for local reactions at the injection site.

Twenty-two days after the second immunization, the anesthetized mice were challenged intranasally (i-n.) In a laboratory.

BL4-containing laboratory (P4-Jean Merieux-lNSERM, Lyon, France) with 4.09 x 106 50% Infective dose of influenza A cell culture (CCID50) / Turkey / 582/06 (kindly provided by Dr.

Bruno Lina, Lyon University, Lyon, France). Following the challenge, the mice were observed 5 for all clinical symptoms and weighed daily, over a period of fourteen days.

Mice with severe infection symptoms and> 25% weight loss were euthanized after anesthesia.

Sanctuary collection, lung and nasal lavage and spleen collection Collection of saphenous vein blood was performed fourteen days 10 after the first immunization and fourteen days after the second immunization in the non-anesthetized animal.

The serum was collected by centrifugation at 8000 g for 10 min.

Four weeks after the second immunization, the mice were anesthetized with CO2 gas and immediately after termination, cardiac puncture was used to collect blood.

After final bleeding, a catheter was inserted into the trachea in the lungs and one ml of cold PBS-protease inhibitor cocktail solution was placed in a lcc syringe attached to the catheter and injected. into the lungs and then removed for analysis.

This washing procedure was performed twice.

The lung washes were centrifuged to remove cell debris.

For nasal washes, a catheter was inserted into the nasal area and 0.5 ml of the PBS-protease inhibitor cocktail solution was pulled through the catheter into the nasal passages and then collected.

Nasal washes were centrifuged to remove cell debris.

Spleen collection was performed in mice immunized intramuscularly with 5 µg of vaccine produced from pIanta with adjuvant, or 5 µg of recombinant H5 antigen with adjuvant, as well as in mice immunized intranasally with 1 µg of cavine produced from plant with adjuvant, or 1 µg of recombinant H5 antigen with adjuvant.

The connected spleens were placed in RPMI supplemented with 30 gentamycin and mixed in a 50 ml conical tube with a 10 ml syringe plunger.

The mixed spleens were rinsed 2 times and centrifuged at 2000 rpm for 5 min and resuspended in

ACK for 5 min at room temperature.

The splenocytes were washed in PBS-gentamycin, resuspended in 5 ° / 0 RPMI and counted.

Splenocytes were used for proliferation assay.

Antibody titrations 5 Antibody serum antibody titers were measured 14 days after the first immunization, as well as 14 and 28 days after the second immunization.

Titration was determined by enzyme-linked immunosorbent assay (ELISA) using the inactive virus A / Indonesia / 5/05 as the coating antigen.

Endpoint titrations were expressed 10 as the reciprocal value of the highest dilution that reached an OD value of at least 0.1 higher than that of negative control samples.

For the determination of the antibody class (lgGl, lgG2a, lgG2b, lgG3, lgM), the titrations were evaluated by ELISA as previously described. 15 Hemaqglutination inhibition titrations (Hl) The serum hemagglutination inhibition (Hl) titrations were measured at 14 and 28 days after the second immunization as above -. described (WHO 2002; Kendal 1982). Inactivated virus preparations from strains A / Indonesia / 5/05 or A / Vietnam / 1203/2004 were used to test 20 mouse serum samples for H1 activity. The sera were pretreated with receptor destruction enzyme ll (RDE ll) (Denka Seiken Co., Tokyo, Japan) prepared from Vibrio cho / erae (Kendal 1982). The H1 assays were performed with 0.5% red turkey blood cells.

Hl antibody titers were defined as the reciprocal of the highest dilution causing complete inhibition of agglutination.

Examples Example 1: Transient expression of qripe A virus / Indonesia / 5/05 (H5N1) hemaqglutinin by aqroinfiltração in N. benthamiana plants The ability of the transient expression system to produce 30 influenza hemagglutinin was determined by the expression of the H5 subtype of strain A / Indonesia / 5/05 (H5N1). As shown in Figure 11, the hemagglutinin (Ace.

No. EF541394),

with its native signal peptide and transmembrane domain, it was first assembled on the plastocyanin expression cassette - promoter, 5'UTR, 3'UTR and transcription termination sequences from the plastocyanine alfalfa gene - and the assembled cassette (660 ) was inserted into a pCAMBIA binary plasmid. The plasmid was then transfected into Agrobacterium (AGLI), creating the recombinant strain AGL1 / 660, which was used for transient expression. N. benthamiana tapirs were infiltrated with AGL1 / 660, and the leaves were collected after a six-day incubation period. To determine whether H5 accumulated in the agro-infiltrated leaves, the protein was first extradited from infiltrated leaf tissue and analyzed by Western blotting using polyclonal anti-H5 antibodies (Vietnam). A single range of approximately 72 kDa was detected in extracts (Figure 12), corresponding to the size for the non-cleaved HAO form of influenza hemagglutinin. The commercial 15 H5 used as a positive control (A / Vietnam / 1203/2004; Protein Sci- ence Corp., Meriden, CT, USA) was detected as two bands of approx.

P uly 48 and 28 kDa, corresponding to the molecular weight of HAI and HA2 fragments, respectively. This demonstrates that H5 expression in infiltrated leaves results in the accumulation of non-cleaved translation product. 20 The formation of active HA trimers was demonstrated by the ability of crude protein extracts from sawdust transformed by AGLI / 660 to agglutinate red turkey blood cells (data not shown). Example 2: Characterization of structures containing hemaqglutinin in plant extracts using size 25 exclusion chromatography The set of hemagglutinin produced by an influenza plant in high molecular weight structures was evaluated by gel filtration. Crude protein extracts from plants infiltrated with AGLI / 660 (1.5 ml) were fractionated by size exclusion chromatography (SEC) on columns of Sephacryi® S-500 HR (GE Healthcare Bio-Science Corp., Piscataway, NJ, U-30 SA). Elution fractions were assayed for their total protein content and for HA abundance using immunodetection with anti-HA antibodies (Figure 13A). As shown in Figure 13A, elution of Blue Dextran (2

MDa) at an anterior peak in fraction 10 while the host protein mass was retained in the column and eluted between fractions 14 and 22. When 200 µL proteins from each SEC elution fraction were concentrated (5 times) by acetone precipitation and analyzed by Western blotting (Figure 15A, 5 H5), hemagglutinin (H5) was mainly found in fractions 9 to 14 (Figure 13B). Without wishing to be bound by theory, this suggests that the HA protein had either mounted on a large superstructure, or that it had attached to a high molecular weight structure.

A second expression cassette was assembled with the Hl nucleic acid sequence from A / New Caiedonia / 20/99 (H1N1) (SEQ ID NO: 33; Figure 16; Gen8ank Accession No.

AY289929) to produce 540 construction (Figure 11). A chimeric gene construct was designed to produce a soluble trimeric form of H1 in which the signal peptide originated from a plant protein gene disulfide isomerase, and the Hl transmembrane domain was replaced by the pll variant of the zipper " of leucine GCN4, a peptide shown to self-assemble in trimer (Harbury et al., 1993) (cassette 544, figure 11). Although lacking the transmembrane domain, this soluble trimeric form was capable of hemagglutination (data not shown) 20 Protein extracts from plants infiltrated with AGL1 / 540 or AGL1 / 544 were fractionated by SEC and the presence of eluted Hl fractions was examined by Western blotting with anti-influenza A antibodies (Fitzgerald, Concord, MA, USA). infiltrated leaves of AGLI / 540, Hl accumulated mainly as a very high molecular weight structure, with peak 25 inclined to smaller size structures (Hl; Figure 13C). In infiitrated sheets of AGLI / 544, the shape H soluble l accumulated as isolated trims as demonstrated by the gel filtration elution model that is parallel to the host protein elution profile (soluble Hl; Figure 13D). In comparison, H1 shells (Protein Science Corp., Me-30 riden, CT, USA), consisting of 5-6 trimester hemagglutinin micelles eluted in fractions 12 to 16 (Figure 13E), earlier than the soluble form H1 (Figure 13D) and later than native H1 (Figure 13C).

To assess the impact of coexpression of Ml in hemagglutinin array in structure, an Ml expression cassette was assembled using nucleic acid corresponding to the coding sequence of A / PR / 8/34 (H1N1) Ml (SEQ ID NO: 35; Figure 18; Gen8ank Accession No. 5 NC 002016). The construction was named 750 and is shown in Figure

11. For the coexpression of Ml and Hl, suspensions of AGL1 / 540 and AGL1 / 750 were mixed in an equal volume before infiltration. Co-infiltration of multiple Agrobacterium suspensions allows coexpression of multiple transgene. Western blot analysis of elution SEC fractions shows that Ml coexpression does not change the elution profile of H1 structures, but has resulted in a decrease in H1 accumulation in agro-filtered leaves (see Figure 13F). Example 3: Isolation of H5 structures by centrifugation in sucrose qradient and observation under electron microscopy Observation of the hemagglutinin structure under electron microscopy (EM) required a higher concentration and purity level than that obtained from SEC in extracts of raw leaf protein. To allow EM observation of H5 structures, a raw leaf protein extract was first concentrated by precipitation of PEG (20% PEG), followed by resuspension in 1/10 volumes of extraction buffer. The concentrated protein extract was fractionated by S-500 HR gel filtration and elution fractions 9, 10, and 11 (corresponding to the column void volume) were pooled and additionally isolated from host proteins by ultracentrifuge in a 20-60 ° 6 sucrose density gradient. The sucrose gradient was fractionated starting from the top, and the fractions were dialyzed and concentrated in a 100 NMWL centrifugal filter unit before analysis. As shown in Western blots and hemagglutination results (Figure 14A), H5 accumulated mainly in fractions 16 to 19 that contained = 60 ° /, sucrose, so many of the host proteins in fractions at peak 13. Fractions 17, 18, and 19 were collected, negatively stained, and observed under MS examination. of the sample that clearly demonstrated the presence of perforated spherical structures varying in size from 80 to 300 strokes that match the morphological characteristics of influenza VLPS (Figure 14B). Example 4: Purification of VLPS H5 from Plant Biomass Flu In addition to an abundant content of soluble proteins, plant leaf extracts 5 contain a complex mixture of soluble sugars, nucleic acids and lipids.

The crude extract was cleared by a change in pH and heat treatment, followed by filtration on diatomaceous earth (see Material and method section for a detailed description of the bleaching method). Figure 15A (lanes 1-4) shows a complete protein content stopping Coomassie Blue stained gel at the various stages of seaming.

A comparison of protein content in the crude extract (lane 1) and in the bleached extract (lane 4) reveals the ability of the bleaching steps to reduce the overall protein content and remove much of the largest visible contaminant in 50 kDa in raw leaf extracts.

The 50 kDa range corresponds to 15 large Ru8isCO subunits, representing up to 3 3 ° / o of total leaf proteins.

The H5 VLPS of influenza was purified from these bleached extracts. by affinity chromatography on a fetuin column.

A comparison of the load fraction (Figure 15A, lane 5) with the flow through (Figure 15A, lane 20 6) and the eluted VLPS (Figure 15A, lane 7) demonstrate the specificity of the fetuin affinity column for H5 Flu VLPs in lightened plant extract.

The purification procedure resulted in more than 75% purity in H5, as determined by densitometry in the Coomassie 25 Blue stained SDS-PAGE gel (Figure 15A, lane 7). In order to assess the structural quality of the purified product, the purified H5 was concentrated in a centrifugal filter unit of 100 NMWL (Limit of nominal molecular weight) and examined under MS after negative staining.

Figure 15B shows a representative sector showing the presence of profuse VLPS.

A closer examination confirmed the presence of peaks in the VLPS (figure 15C). As shown in Figure 15D, H5 VLPS were purified to approximately 89% purity of bleached leaf extract by chromatography.

affinity matography in a fetuin column, based on the density of the spotted Coomassie Blue H5 hemagglutinin and on the determination of the total protein content by the BCA method. HA VLPS bioactivity was confirmed by its ability to agglutinate red turkey blood cells (data not shown). Figure 20B also confirms the identity of the purified VLP visualized by Western blotting and immunodetection with a polyclonal anti-H5 serum (A / Vietnam / 1203/2004). A single band of approximately 72 kDa is detected and corresponds in size to the form of non-cleaved HAO of flu heglutinin. Figure 15C shows the VLP structure of the vaccine with hemagglutinin peaks covering its structure. VLPS were formulated for immunization of mice by filtration through a 0.22 µm filter; endotoxin content was measured during an LAL endotoxin detection kit (Limulus Amebocyte Lysate) 15 (Lonza, Walkserville, MS, USA). The filtered vaccine contains 105.8 ± 11.6% EU / ml (endotoxin units / ml).

+ Example 5: Localization of Flu VLPS in plants To locate VLPs and confirm their plasma membrane origin, sections of thin leaves from H5 production plants were fixed and examined under TEM after positive staining. The observation of leaf cells indicates the presence of VLPS in extracellular cavities formed by the invagination of the plasma membrane (Figure 19). The shape and position of the observed VLPS demonstrate that despite the affixing of their plasma membranes to the cell wall, plant cells have the plasticity required to produce influenza VLPS derived from their plasma membrane and accumulate it in the apoplastic space. . Example 6: Plasma membrane lipid analysis In addition, confirmation of the composition and origin of the pIanta flu VLPS was obtained from analysis of the lipid content. The lipids were extracted from purified VLPS and their composition was compared to that of highly purified tobacco plasma membranes by performance thin layer chromatography (HP-TLC). The polar and neutral lipid migration models of VLPS and plasma membrane control were similar.

The purified VLPS contained the largest phospholipids (phosphatidylcholine and phosphatidylefhanolamine) and sphingolipids (glucosylceramide) found in the plasma membrane (Figure 27A), and both contained free sterols as the neutral lipids only (Figure 27B). However, the immunodetection of a plasma membrane protein marker (ATPase) in purified VLP extracts showed that the VLP lipid bilayer does not contain one of the largest proteins associated with plant plasma membranes, suggesting that proteins hosts may have been excluded from membranes during the process of origin of VLPs from plant cells (Figure 27C). Example 7: Immunogenicity of H5 VLPS and effect of route of administration The mice were administered with H5 VLPs produced per plant by intramuscular injection, or intranasal (inhalation). 0.1 to 12 µg of 15 VLPs were injected intramuscularly into the mice, with alum. as an adjuvant, according to the methods described.

Peak antibody titers were observed with the lowest antigen amount. xa, in a quantity similar to that of 5 µg of soluble hemagglutinin recombinant (HA) (Figure 20A). 20 0.1 to 1 µg of H5 VLPS produced from the plant were administered intranasally with a chitosan adjuvant provided for an antibody response greater than that of recombinant soluble HA with an alum adjuvant (Figure 20B). For both routes of administration, and over a range of 25 amounts of antigen, seroconversion was observed in all mice tested.

The recombinant H5 soluble antigen conferred low ("1/40) or reasonable (1" 1/10 for non-adjuvant recombinant H5) H1 titrations. Example 8: Titration inhibition antibody (HAI) H5 30 VLP Figure 21 A, B illustrates the response of hemagglutination inhibition antibody (HAI) 14 days after a "boost" with H5 VLP pro-

from plant, or recombinant soluble HA. The lowest dose of antigen (0.1 µg) when administered intramuscularly produced a higher HAI response for administration greater than 10 times (5 µg) of recombinant soluble HA. Increased doses of H5 VLP provided a modest increase in HAI over the lowest dose. The HAI response following intranasal administration was significantly increased in mice administered with H5 VLPS produced from plant (1.0 or 0.1 µg) compared to those administered with 1 µg of recombinant soluble HA, which was similar to the negative control. All 10 mice immunized by intramuscular injection of H5 VLPS (from 0.1 to 12 µg) had higher HAI titers than mice immunized with the control HA antigen (Figure 4a - now 21 A). For the same dose of 5 µg, VLPS induced HAI titrations 20 times higher than the corresponding dose of HA control antigen. VLPS 15 also induced HAI titrations significantly higher than the control HA antigen when distributed via the intranasal route (Figure 21b). For a given dose of H5 VLP, HAI titration levels

H were lower in mice immunized intranasally than for mice immunized intramuscularly; 1 µg of VLP induced an average HAI titration of 210 when administered i.m. while the same dose induced an average HAI titration of 34 administered i.n .. When administered intramuscularly, all doses of VLPS induced a high level of antibodies capable of binding homologous to the total inactivated viruses (Figures 20b and 24). No significant difference was found between the plant-produced VLP vaccine and the control HA antigen (except the 12 µg VLP group 14 days after impulse), as both antigen preparations induce high binding of antibody titers against the homologous strain. However, when administered intranasally, VLPS induced higher binding of antibody titers than the antigen of contrasting HA (Figure 20b). When mixed with Chitosan, immunization with a microgram of VLP induced a reciprocal mean Ab titration of 5 500, 8.6 times higher than the level found in mice immunized with 1 µg of the HA control antigen ( reciprocal average Ab titre of 920). The immunogenicity of the plant-derived influenza VLPS was then investigated through a dose variation study in mice. Groups of five B ALB / C mice were immunized intramuscularly twice at 3-week intervals with 0.1 µg to 12 µg HA containing VLPS of Influenza A / Indonesia / 5/05 (H5N1) formulated in alum (ratio 1: 1). The hemagglutination inhibition (Hl) titrations, using total inactivated virus antigen (A / Indonesia / 5/05 (H5N1)), were measured in the serum collected 14 days after the second immunization. Immunization with doses of VLP lower than 0.1 µg induced the production of antibodies that inhibited agglutination viruses and erythrocytes at high dilutions (Figure 21 A). Parallel immunization of mice with 5 µg of control H5 antigen with non-VLP adjuvant alum (also from A / Indonesia / 5/05) 15 induced an Hl response that was 2-3 logs lower than that- "tired with the lowest VLP dose.

R For both routes of administration, and over a range of antigen quantities, the HAI response is superior in mice administered with VLPS. 20 Example 9: Effect of adjuvant on the immunosuppression of H5 VLPs The H5 VLPS produced from plants have a plasma membrane origin (Figure 19, Example 5). Without wishing to be bound by theory, enveloped viruses or enveloped virus VLPs usually acquire their envelope from the membrane they originate from. Plasma membranes from plants have a phytosterol supplement that is rarely, if ever found in animal cells, and several of these sterols have been shown to exhibit immunostimulatory effects. H5 VLPS produced from plants were administered intramuscularly (Figure 22A) or intranasally (Figure 22B) to mice in the presence or absence of an adjuvant, and HAI (hemagglutination inhibiting antibody response) determined. VLPS, in the presence or absence of an added adjuvant (alum or chitosan, as in these examples) in any delivery system demonstrated a significantly greater inhibition of hemaglutinin HAI than recombinant soluble HA.

Even in the absence of an added adjuvant (i.e., alum or chitosan), the H5 VLPS produced from the plant demonstrate a significant HAI, indicative of a systemic immune response to administration of the antigen.

The alum increased the mean level of HAI titrations by a factor of 5 for intramuscular administration of VLP (Figure 22a), and by a factor of 3.7 for the control HA antigen.

When administered i.m., 5 µg of VLPs induced an average HAI titration 12 times higher than 10 than the corresponding control HA antigen dose.

Chitosan does not boost the average HAI level of the control HA antigen (Figure 22b), while increasing the average HAI level of mice immunized with 1 µg of VLP administered i.n. by a factor of 5 times.

Example 10: lsotype of antibody 15 Mice administered with H5 VLPS produced from plant or recombinant soluble HA in the presence or absence of alum as an added adjuvant demonstrated a variety of immunoglobulin isotypes. (Figure 23 A). In the presence of an added adjuvant, the VLPS and HA antibody isotype profiles are similar, with IgGl being the dominant isotype.

When VLPS or HA are administered without an added adjuvant, the IgGl response is reduced, but the dominant isotype response for VLPS remains, with lgM, lgG2a, lgG28 and lgG3 maintaining similar titrations as in the presence of an added adjuvant.

Titrations of IgGl, IgG2a, and 25 IgG2b are markedly reduced when HA is administered without an adjuvant added.

These data, therefore, demonstrate that VLPS produced from pIanta do not require an added adjuvant to induce an antibody response in a host. 30 Antibody titers against total inactive influenza virus strains (A / Indonesia / 5/05; A / Vietnam / 1203/04) 1 in mice administered with VLPS produced from plant or recombinant soluble HA

muscle in the presence of an added antigen are illustrated in Figure 23B. No significant difference is observed in the antibody titers for these flu strains in mice administered with 1 µgou5µgdeVLPsou5µgdeHAsoluble. 5 Example 11: Cross reactivity of serum antibodies induced by the H5 VLP vaccine Cross reactivity of serum antibodies induced by H5 VLP was evaluated against total inactivated influenza viruses of different strains. All doses of VLP (from 0.1 to 12 µg), as well as 5 µg of control HA 10 antigen induced high binding of antibody titers against a strain 1 strain (A / Vietnam / 1194/04) , the homologous strain A / Indonesia / 5/05 of clade 2.1, and a clade strain 2.2 A / turkey / Turkey / 1/05 (Figure 25A). However, only the VLP produced from pIanta induced HAI titration against the A / turkey / 1 "urkey / 1/05 strain (Figure 25b). The HAI titrations for A / Indonesia / 5/05 were high for VLPS Example 12: Cross protection provided by immunization with H5 VLP pro-

Plant production Mice that had previously been administered a two-dose regimen of A / Indonesia / 5/05 H5 VLPS as described above, were subsequently challenged intranasally with ATurkey Influenza virus / 582/06 (H5N1) ("Turkey H5N1"), and observed. The dose administered, per animal, was 10 LD50 (4.09 X 10 'CClD50). For 7 days post-challenge, only 37.5% of the mice administered 25 with the PBS vaccine control had survived exposure to H5N1 turkey (Figure 26A). 100 ° / o of the animals administered with the control antigen (HA) or 1, 5 or 15 µg of Indonesian H5 VLPS survived up to 17 days post-challenge, when the experiment was completed. The body mass of the mice was also monitored 30 during the experiment, and the average mass of the surviving mice plotted (Figure 26B). Mice administered with 1, 5 or 15 µg of the Indonesian H5 VLPS prior to the challenge did not lose any appreciable mass during the course of the experiment, and, in particular, mice administered with 5 µg of the VLPS appeared to have gained significant mass. The negative control mice (no challenge from Turkey H5N1) did not appreciably gain or lose body mass. The 5 positive control mice (not administered with VLPs, but challenged with Turkey H5N1) exhibited significant loss of body mass during the course of the experiment, and three of these mice died. As body mass is an average of all mice in the group, removal of the 'sickest' mice (the 3 who died) can lead to an apparent total increase in mass, however it is noted that body mass The mean area of the positive control group is still significantly below that of the negative or VLP treated groups. These data, therefore, demonstrate that influenza VLPS produced from plants comprising the viral protein H5 hemagglutinin induce a specific immune response to pathogenic influenza strains, and that virus-like particles can originate from a plasma membrane of plant.

V These data, therefore, demonstrate that plants are capable of producing influenza virus-like particles, and also for the first time, that virus-like particles can originate from a plant plasma membrane. In addition, using current transient expression technology, a first batch of antigen was produced only 16 days after the target HA sequence was obtained. Under current yields for H5 VLPS, 25 and at an exemplary dose of 5 µg per individual, each kg of infiltrated leaf can produce -20,000 doses of vaccine. This unique combination of platform simplicity increases capacity and provides powerful immunogenicity for, eritre other embodiments, a new method response in the context of a pandemic. 30 Example 13: Characterization of structures containing hemaqglutinin in plant extracts using size exclusion chromatography The set of influenza hemagglutinin produced by plants of different subtypes in high molecular weight structures was evaluated by gel filtration.

Crude or concentrated protein extracts from infiltrated plants of AGL1 / 660-, AGL1 / 540-, AGL1 / 783-, AGL1 / 780- and AGLI / 785 (1.5 ml) were fractionated by size exclusion chromatography ( SEC) in 5 columns of Sephacryl® S-500 HR (GE Healthcare Bio-Science Corp., Pistaway, NJ, USA). As shown in Figure 46, Blue Dextran (2 MDa) with anterior peak elution in fraction 10. When the 200 µL proteins from each SEC elution fraction were concentrated (5 times) by acetone precipitation and analyzed by Western blotting (Figure 46), hemaglolO tinins were mainly found in fractions 7 to 14, and are indicative of the incorporation of HA in VLPs.

Without wishing to be bound by theory, this suggests that the HA protein had either mounted on a large superstructure or that it had attached to a high molecular weight structure, indifferent to the subtype produced. 15 Example 14: Transient expression of hemaqglutinin from the seasonal qripe virus by anro-infiltration in N. benthamiana plants The ability of the transient expression system to produce he-. seasonal influenza maglutinin was determined by expressing the Hl subtype of strains from A / 8risbane / 59/2007 (H1N1) (plasmid N ° 774), A / New 20 Caledonia / 20/1999 (H1N1) (plasmid N ° 540) and A / Ilhas Salomão / 3/2006 (H1N1) (plasmid No. 775). The hemagglutinin gene coding sequences were first assembled on the plastocyanin expression cassette - promoter, 5'UTR, 3'UTR and transcription termination sequences from the plastocyanine alfalfa gene - and the assembled cassettes were inserted 25 in a binary plasmid from pCAMBIA.

The pIasmids were then transfected into Agrobacterium (AGLI), producing strains of Agrobacterium AGLI / 774, AGLI / 540 and AGL1 / 775, respectively.

N. benthamiana plants were infiltrated with AGL1 / 774, AGL1 / 540 and AGL1 / 775, and the leaves were collected after a period of six 30 days.

To determine whether H1 accumulated in the agro-infiltrated leaves, the protein was first extracted from infiltrated leaf tissue and analyzed by Western blotting using anti-H1 antibodies. A single band of approximately 72 kDa was detected in extracts (Figure 47), corresponding in size to the form of non-cleaved HAO of influenza hemagglutinin. This demonstrates that expression of annual epidemic strains other than hemagglutinin in infiltrated leaves results in the accumulation of non-cleaved translation product. 5 Example 15: Transient expression of potential pan-viral qripe hemagglutinin by water infiltration in N. benthamiana plants The ability of the transient expression system to produce potential influenza hemagglutinin was determined by expressing the H5 subtype of A / strains Anhui / 1/2005 (H5N1) (plasmid No. 781), 10 A / Indonesia / 5/2005 (H5N1) (plasmid No. 660) and A / Vietnam / 1194/2004 (H5N1) (plasmid # 782). The hemaglutinin gene coding sequences were first assembled on the pIastocyanin expression cassette - promoter, 5'UTR, 3'UTR and transcription termination sequences from the plastocyanine alfalfa gene - and the assembled cassettes were inserted 15 in a binary plasmid from pCAMBIA. The plasmids were then transfected into Agrobacterium (AGLI).

V Plants of N. benthamiana were infiltrated with AGL1 / 781, AGL1 / 660 and AGL1 / 782, and the leaves were collected after a period of six days. To determine whether H5 accumulated in the agro-infiltrated leaves, the protein was first extracted from infiltrated leaf tissue and analyzed by Western blotting using anti-H5 antibodies. A single band of approximately 72 kDa was detected in the extracts (Figure 48), corresponding in size to the non-cleaved HAO form of influenza hemagglutinin. This demonstrates that expression of potential pandemic strains other than hemagglutinin in 25 infiltrated leaves results in the accumulation of non-cleaved translation product. Example 16: Transient expression of H5 by water infiltration in plants of N. tabacum The ability of the transient expression system to produce influenza hemagglutinin in leaves of Nicotiana tabacum was analyzed using the expression of the H5 subtype of A / Indonesia / 5/2005 (H5N1) (plasmid No. 660). The hemagglutinin gene sequences were first assembled on the plastocyanin expression cassette - promoter, 5'UTR,

3'UTR and transcription termination sequences from the alfalfa plastocyanin gene - and the assembled cassettes were inserted into pCAMBIA binary plasmid.

The plasmids were then transfected into Agrobacterium (AGLI). 5 Nests of N. tabacum were infiltrated with AGL1 / 660 and the leaves were collected after a period of six days.

To determine whether H5 accumulated in the agro-infiltrated leaves, the protein was first extracted from infiltrated leaf tissue and analyzed by Western blotting using anti-H5 antibodies. A single band of approximately 72 kDa was detected in the extracts (Figure 49), corresponding in size to the non-cleaved HAO form of influenza hemagglutinin. This demonstrates that hemagglutinin expression in infiltrated N. tabacum leaves results in the accumulation of non-cleaved translation product.

Example 17: Immunogenicity of H5N1 VLP vaccine produced by plant 15 of A / Indonesia / 5/05 (H5N1) in ferrets A dose scale study in ferrets was performed to assess

4 ar the immunogenicity of plant-derived VLPS.

In vitro reactivity of serum antibody that induces the H5 VLP vaccine in 3 doses (1.5 and 15 µg) was evaluated by inhibiting the hemagglutination of three other strains of 20 H5N1 - A / turkey / Peru / 1/05 ( 2.2), A / Vietnam / 1194/04 (clade 1) and A / Anhui / 5/05 (all inactivated total viruses), using serum taken 14 days after the first vaccine dose (Figure 50A), and 14 days after the second dose (Figure 50 B). For all 3 dose concentrations, cross-reactivity is observed. Example 17: Analysis of immunogenicity results according to the CHMP criterion.

The EMEA'S Committee for Medicinal Products for Human Use (CHMP) (http: //.emea.europa.eu/htms/general/contacts/CHMP/CHMP.html) adjusts three criteria (then applied the second dose) for vaccine efficacy: 1 —Number of seroconversion or significant increase in H1 titrations (4 times) "40%; 2 - Average geometric increase of at least 2.5; 3 -

proportion of individuals who achieve an Hl titration of 1/40 must be at least 7 ° / o.

The analysis of these criteria in the ferret model is shown in Tables 8-11. (*) is indicative of meeting or exceeding the CHMP criteria.

A summary of cross-immunogenicity analysis against 5 the CHMP criteria for licensing is shown in Table 12. The animals were assessed daily for body weight, temperature and total condition.

No signs of illness or discomfort were recorded during the study.

Body weight and temperature were within normal ranges during the study.

The vaccine was safe and tolerated by the study animals.

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Strain Criterion Study group With ad- 5µg with 15 µg with adjuvant adjuvant adjuvant

A / turkey / Turkey / 1 / ° /, 4-fold increase in titu- 8Oq / o * 100 ° 6 * 80O / j *

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Example 18: Selection of hamaqglutinin nucleotide sequences HA nucleotide sequences were retrieved from a Flu sequence database (see URL: flu.lanl.qov), or the NCBI source of influenza virus (see URL : ncbi.nmhih.gov/genomes/FLU/FLU.html). For several of the HA nucleic acid sequences, multiple entries are listed in the databases (Table 13). Some variation is mainly associated with the culture system (Origin - MDCK, egg, unknown, viral RNA / clinical isolate); for example, the glycosylation site at position 194 (mature protein numbering) of HA is absent when influenza virus type B is expressed in allantoic fluid from eggs (see also Chen et al., 2008). For some sequences, domains may be lacking (for example, incomplete clones, sequencing artifacts, etc.). The hemagglutinin sequence can be divided into 5 domains: signal peptide (SP), HAI, HA2, transmembrane (DTm) and cytoplasmic tail.

The first sequence domains can be combined with an existing second sequence domain, for example, the signal peptide from a first strain sequence can be combined with the rest of the hemagglutinin coding sequence from a second strain to provide a complete coding sequence.

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- Strain: H1 of A / 8risbane / 59/2007. Position 203 exhibited a variation of aspartic acid (D), isoleu-. kine (I) or asparagine (N). 15 Cepa: H3from AJ8risbane / 10/2007 Sequence variations were observed in the 5 positions (Table 15). At position 215, an override is observed in two sampled sequences.

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EU199366 Egg GPASI ISDN273757 Egg VL-SS a

ISDN257043 Egg GPASl EU199250 MDCK GLASI ISDN375357 T Egg ISDN260430 I Egg I GPASl

Origin 202, 210, 215, 235 242 * ISDN256751 Egg GPASl ISDN257648 MDCK GLASI * Numbering from the beginning M Strain: H3 for A / Wisconsin / 67/2005 Sequence variations in this strain were observed in the 4 positions (Table 16) . 5 Table 16: H / A / Wisconsin / 67/2005 amino acid variation Origin 138, 156, 186, 196 ISDN138724 Unknown AHGH DQ865947 Unknown SHVY EF473424 Unknown AHGH ISDN138723 Egg SQVY ISDN131464 Unknown AHGH EF473455 'Egg' HH * of mature protein r Cepa: from B / Malaysia / 2506/2004

E Variation in two positions is observed (Table 17). Position 120 is not a glycosylation site; position 210 is involved in glycosylation; 10 this glycosylation is then abolished from egg culture. Table 17: B hemaqlutinin / Malaysia / 2506/2004 amino acid variation Amino acid #: MDCK Egg 120 K N 210 T A * Numbering from the intermediate part of SP Cepa: hemaqglutinin from B / Florida / 4/2006; ISDN261649 Observed variations include sequence variation of amino- "15 acid at position 211, depending on the culture system. Asparatin (N) is found in isolated sequences of MDCK cells, while acid

Glutamic W (D) is found in an isolated sequence of eggs. Position 211 is a glycosylation site, and egg culture is then abolished. Strain: H2 from A / Sinqapura / '1/1957 20 Variations of sequence were observed in the 6 positions (

beautiful 18). Table 18: H2 of amino acid variation of A / Sinqapura / 1/1957 Origin Amino Acid No. 166 168 199 \ 236 238 358 L20410 Viral RNA KETLSV Ll1142 Unknown EGKLSI AB296074 Unknown KGTQGV Consensus KGTQ / LGV I A / Japan / 305/1957 i

1 Numbering from the mature protein Strains: H5 from AJVietnam / 1194/2004 and H5from A / Anhui / 1/2005 5 There are no variations observed in the amino acid sequence under alignment of the primary sequences of any of these H5 strains. Strain: H6 of A / Teal / Honçj Konq / W312 / 1 997 Only one entry was available for strain (AF250179). Strain: H7 for A / Equine / Praçja / 56 "10 A total of 2 sequence entries were found in

- data base.

The entry AB298877 was excluded, since it is a laboratory "sortant".

Strain: H9 of A / Honçj Konçj / 1073/1999; AJ404626 A total of 2 sequence entries were found in the 15 databases.

Only one was complete.

All citations are thus incorporated by reference.

The present invention has been described with respect to one or more embodiments.

However, it will be apparent to those skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Reference List: Aymard, H.

M, M. T.

Coleman, W.

R.

Dowdle, W.

G.

Laver, G.

Ç.

Schild, and R.

G.

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Claims (15)

1. Nucleic acid comprising a nucleotide sequence that encodes an influenza hemagglutinin (HA) operably linked to an active regulatory region in a plant. Nucleic acid according to claim 1, in which influenza hemagglutinin is selected from the group consisting of Hl, H 2, H3, H4, H5, H6, H7, H8, H9, HlO, H11, H12, H13, H14, H15, and H16. 3. A method of producing influenza virus-like particles (VLPS) in a plant comprising: a) introducing the nucleic acid as defined in claim 1 into the plant, or portion thereof, and b) incubating the plant under conditions that allow expression nucleic acid, thereby producing VLPS. Method according to claim 3, in which in the introduction step (step a), the nucleic acid is transiently expressed in the pIanta. - Method according to claim 3, in which in the introduction step (step a), the nucleic acid is stably expressed in the plant. . 6. Method according to claim 3, additionally comprising the step of 20 C) collection from the host and purification of the VLPS. 7. Virus-like particle (VLP) comprising an influenza virus HA protein and one or more than one lipid derived from a pIanta. 8. Virus-like particulate (VLP) according to claim 257, in which the influenza HA protein is Indonesian H5. A composition comprising an effective dose of VLP as defined in claim 7, and a pharmaceutically acceptable carrier. A method of inducing immunity to an influenza virus infection in an individual, comprising administering the virus-like particle 30 as defined in claim 7. A method according to claim 10, in which the virus-like particle is administered to an individual orally, intradermally, Ç) intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. 12. Virus-like particulate (VLP) comprising an HA flu virus bearing plant-specific N-glycans, or modified N-glycans. A composition comprising an effective dose of the VLP according to claim 12, and a pharmaceutically acceptable carrier. 14. Method of inducing immunity to an influenza virus infection in an individual, comprising administering the composition according to claim 13. A method according to claim 11, in which the composition is administered to an individual orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.