WO2011011390A1 - Purified recombinant influenza virus ha proteins - Google Patents

Abstract

Disclosed is a purified viral protein and/or VLPs with high purity and high vaccine potency, and methods of purifying thereof Further disclosed are uses of the purified viral protein and/or VLPs as a standard in a vaccine potency assay

Description

PURIFIED RECOMBINANT INFLUENZA VIRUS HA PROTEINS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 61/226,994, filed July 20, 2009, which is herein incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] This invention relates to generally to the purification of influenza HA proteins and to methods of use thereof.

BACKGROUND OF INVENTION

[0003] Influenza virus is a member of Orthomyxoviridae family (for review, see Murphy and Webster, 1996). There are three subtypes of influenza viruses designated A, B, and C. The influenza virion contains a segmented negative-sense RNA genome. The influenza virion includes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PBl). polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2) proteins. The HA, NA Ml, and M2 are membrane associated, whereas NP. PBl, PB2_> PA, and NS2 are nucleocapsid associated proteins. The NSl is the only nonstructural protein not associated with virion particles but specific for influenza-infected cells. The M 1 protein is the most abundant protein in influenza particles. The HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the ceil, and the sources of the major immunodominant epitopes for virus neutralization and protective immunity. Both HA and NA proteins are considered the most important components for prophylactic influenza vaccines because they are highly immunogenic.

[0004] Influenza virus infection is initiated by the attachment of the virion surface HA protein to a sialic acid-containing cellular receptor (glycoproteins and glycolipids). The NA protein mediates processing of the sialic acid receptor, and virus penetration into the cell depends on HA-dependent receptor-mediated endocytosis. In the acidic confines of internalized endosomes containing an influenza virion, the HA protein undergoes conformational changes that lead to fusion of viral and host cell membranes followed by virus uncoating and M2-mediated release of Ml proteins from nucleocapsid-associated ribonucleoproteins (RNPs), which migrate into the cell nucleus for viral RNA synthesis. Antibodies to HA molecule can prevent virus infection by neutralizing virus infectivity, whereas antibodies to NA proteins mediate their effect on the early steps of viral replication.

[0005] Prior art processes for producing recombinant HA can generate HA proteins with conformational structures that are not recognized by the immunized host as native, resulting in poor antigenicity in assays such as the single radial immunodiffusion assay (SRlD assay). The present inventors have developed improved purification methods that produce purified HA proteins with surprising and unexpected antigenic properties. When administered to a host animal such as a sheep, the purified HA proteins are recognized as native and the host animal produces antibodies that recognize the virus. In contrast, prior art methods produce denatured HA proteins that are poorly antigenic in a host animal.

SUMMARY OF THE INVENTION

[0006] The present invention provides purified HA proteins that demonstrate increased antigenicity as compared to prior art HA proteins.

[0007] In one aspect, the present invention provides a purified viral protein. In one embodiment, the purity of the viral protein is at least about 95%. In another embodiment, the viral protein is a recombinant or chimeric protein derived from an influenza virus. In some embodiments, the influenza virus is selected from the group consisting of avian influenza virus and mammalian influenza virus. In one embodiment, the mammalian influenza virus is a human influenza virus strain. In an alternative embodiment, the mammalian influenza virus is a swine influenza virus. As described herein, said viral protein may be derived from a pandemic strain or a seasonal strain of influenza virus.

[0008] In one embodiment, the purified viral protein is an HA protein. In some embodiments, the HA protein is selected from the group consisting of Hl, H2, H3, H4, H5, Hό, 11,7 H8, H9, H10, H11 1, HI2, H13, H 14 , H15 and H16. In an exemplary embodiment, the HA is derived from an HlNl influenza virus strain. In one embodiment, the HlNl influenza virus strain is selected from the group consisting of A/Califomi a/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007. In a preferred embodiment, the purified HA protein exhibits enhanced or improved antigenicity in a single radial immunodiffusion (SRlD) assay as compared to partially denatured or denatured HA proteins. [0009] In another aspect, the present invention provides a method of purifying an HA protein, comprising steps of:

(i) Expressing said HA in a host cell;

(ii) Extracting said HA from said host cell, wherein said HA is soluble in an extraction buffer;

(iii) Purifying said HA from an extraction buffer using ion exchange chromatography, wherein a pool of fractions containing said HA is produced;

(iv) Purifying said HA from said pool in step (iii) using affinity chromatography, wherein a second pool of fractions containing said ItA is produced;

(v) Desalting said second pool in step (iv);

(vi) Purifying said HA from the desalted pool of step (v) using hydroxyapatite chromatography, wherein a third pool of fractions containing said HA is produced; and optionally

(vii) concentrating, dialyzing, and sterilizing said third pool of fractions containing HA.

[0010] In one embodiment, the host cells in step are insect cells. In further embodiments, the insect cells are Sf9 insect cells.

[0011] In one embodiment, the ion exchange chromatography in step (iii) is anion exchange chromatography. In another embodiment, said anion exchange chromatography is a TMAE anion exchange chromatography.

[0012] In one embodiment, the affinity chromatography in step (iv) is a lentil lectin affinity chromatography.

[0013] In one embodiment, the desalting step (v) is conducted with desalting chromatography. In another embodiment, the desalting chromatography is performed using a desalting column packed with Sephadex™ 0-25. In yet another embodiment, the desalting step (v) is conducted with dialysis. In one embodiment, the dialysis is performed using a dialysis bag, tubing, or a stirred cell comprising a selectively permeable membrane. In another embodiment, the selectively permeable membrane has a molecule weight cut off (MWCC)) of about 20 to about 30 KD.

[0014] In one embodiment, the hydroxyapatite chromatography in step (vi) is performed using a column packed with synthetic hydroxyapatite. in further embodiments, the synthetic hydroxyapatite is ceramic hydroxyapatite (e.g., CHT™ hydroxyapatite Type I). [0015] In one embodiment, the viral protein is concentrated and dialyzed in a stirred cell with selectively permeable membrane in step (vii). In another embodiment, the viral protein is steriled by passing through a micron filter (e.g., a 0.2 micron SFCA (or PVDF) filter).

[0016] In another aspect, the present invention provides VLPs comprising a purified HA. In some embodiments, the HA protein is selected from the group consisting of Hl, H2, H3, H4, H5, H6, H,7 H8, H9, HlO, HI l, H12, H13, H14 , H15 and H16. In an exemplary embodiment, the HA is derived from an Hl Nl influenza virus strain. In one embodiment, the HlNl influenza virus strain is selected from the group consisting of A/Ca]ifornia/04/09, A/New Caledonia/20/ 1999, A/Solomon ls/3/2006, and A/Brisbane/59/2007.

[0017] In some embodiments, the VLPs further comprises at least one influenza matrix protein (Ml). In one embodiment, the Ml protein is derived from an HlNl influenza virus strain. In one embodiment, the Hl Nl influenza virus strain is selected from the group consisting of A/Califoraia/04/09, A/New Caledonia/20/ 1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007. In some alternative embodiments, the Ml is derived from an avian influenza virus strain.

[0018] In additional embodiments, the VLPs further comprise at least one influenza neuraminidase protein. In one embodiment, the NA protein is derived from an Hl Nl influenza virus strain. In one embodiment, the H 1 N 1 influenza virus strain is selected from the group consisting of A/California/04/09, A/New Caledonia/20/ 1999, A/Solomon ϊs/3/2006, and A/Brisbane/59/2007.

[0019] In one embodiment, the VLPs further comprise at least one influenza M2 protein. In another embodiment, the VLPs further comprise at least one nucleocapsid (NP) protein.

[0020] The present invention also comprises methods of purifying said VLPs, wherein said methods comprise steps of :

a. concentrating clarifying culture medium containing VLP;

b. purifying concentrated diafiltered VLPs on a discontinuous sucrose gradient;

c. further purifying VLPs by anion exchange chromatography, or isopycnic sucrose cushion centrifugation.

[0021] In one embodiment, said anion exchange chromatography is a -(MAE anion exchange chromatography. In another embodiment, said discontinuous sucrose gradient is a 20% to 60% discontinuous sucrose gradient. In another embodiment, said isopycnic sucrose cushion centrifugation is a about 44% isopycnic sucrose cushion centrifugation.

[0022] The present invention also provides a method of using the purified HA protein and/or purified VLPs comprising a purified HA as a standard in a vaccine potency assay. The potency assay has several applications, and may be useful for industrial firms, governmental institutions (e.g., N1H), non-profit organizations (e.g., WHO) and regulatory bodies (e.g., FDA).

[0023] In one embodiment, the vaccine potency assay is a single radial immunodiffusion (SRID) assay. In one embodiment, a purified RA protein is administered to a host animal. In another embodiment, a purified VLl' comprising a purified HA is administered to a host animal. In an exemplary embodiment, both purified HA protein and purified VLPs comprising a purified HA are administered to a host animal. In preferred embodiments, the host animal mounts an immune response to the purified HA protein and/or purified VLP comprising a purified HA protein. In one embodiment, the host animal produces antibodies against the purified HA protein and/or purified VLP comprising a purified HA protein. In some embodiments, said antibodies are used as a standard in the single radial immunodiffusion (SRlD) assay.

[0024] In another embodiment, the purified HA protein and/or purified VLPs comprising a purified HA are used as a standard in a direct challenge of a vaccinated subject with infectious vims. In one embodiment, the direct challenge of a vaccinated subject with infectious virus is evaluated with percentage of subjects showing infection symptoms, virus titer in the serum, or EDso-

[0025] In another embodiment, the purified HA protein and/or purified VLPs comprising a purified HA are used as a standard in an immunoassay, wherein the immunoassay is selected from the group consisting of radioimmunoassay, enzyme immunoassay, fluorescence polarization immunoassay (FPlA), Kinetic Interaction of Microparticles in solution (KIMS), lateral flow technology, or T-cell responses assay. In further embodiments, monoclonal and/or polyclonal antibodies are utilized in the immunoassay. In further embodiments, said enzyme immunoassay is selected from the group consisting of EMIT, Cloned Enzyme Donor Immunoassay (CEDI), and Enzyme- [.inked Immunosorbent Assay (ELISA).

[0026] In additional aspects, the invention provides immunogenic compositions comprising one or more purified HA proteins as described herein. In one embodiment, the invention provides a micelle comprised of one or more purified HA proteins (e.g. an HA micelle).

[0027] The purified HA proteins may be used for the prevention and/or treatment of influenza infection. Thus, in another aspect, the invention provides a method for eliciting an immune response against influenza. The method involves administering an immunologically effective amount of a composition containing a purified HA protein to a subject, such as a human or animal subject.

[0028] In another aspect, the present invention provides pharmaceutically acceptable vaccine compositions comprising a purified HA protein, an HA micelle comprising a purified

HA protein, or a VLP comprising a purified HA protein.

[0029] In one embodiment, the invention comprises an immunogenic formulation comprising at least one effective dose of a purified HA protein. In another embodiment, the invention comprises an immunogenic formulation comprising at least one effective dose of an

HA micelle comprising a purified HA protein. In yet another embodiment, the invention comprises an immunogenic formulation comprising at least one effective dose of a VLP comprising a purified HA protein.

[0030] In another embodiment, the invention provides for a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine formulations of the invention.

[0031] In another embodiment, the invention provides a method of formulating a vaccine or antigenic composition that induces immunity to an infection or at least one disease symptom thereof to a mammal, comprising adding to the formulation an effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein. In a preferred embodiment, the infection is an influenza infection.

[0032] The purified HA proteins of the invention are useful for preparing compositions that stimulate an immune response that confers immunity or substantial immunity to infectious agents. Thus, in one embodiment, the invention provides a method of inducing immunity to infections or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

[0033] In yet another aspect, the invention provides a method of inducing substantial immunity to an influenza vims infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

[0034] Compositions of the invention can induce substantial immunity in a vertebrate (e.g. a human) when administered to the vertebrate. Thus, in one embodiment, the invention provides a method of inducing substantial immunity to an influenza virus infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA proteinu an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein. In another embodiment, the invention provides a method of vaccinating a mammal against influenza comprising administering to the mammal a protection-inducing amount of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

[0035] In another embodiment, the invention comprises a method of inducing a protective antibody response to an infection or at least one symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

j 0036] In another embodiment, the invention comprises a method of inducing a protective cellular response to an influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein. In another embodiment, the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of an HA micelle comprising a purified HA protein. In yet another embodiment, the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a VLP, wherein the VXP comprises a purified HA protein.

BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1 depicts a TMAE chromatogram (Figure Ia) of purifying HA derived from influenza B/Brisbane/60 and SDS-PAGE gel stained with Coomassie blue, loaded with fractions from the TMAE chromatography (Figure 1 b).

[0038] Figure 2 depicts a lentil lectin chromatogram (Figure 2a) of purifying HA derived from influenza B/Brisbane/60 and SDS PAGB gel stained with Coomassie blue, loaded with fractions from the lentil lectin chromatography (Figure 2b)

[0039] Figure 3 depicts a CHT™ Hydroxyapatite chromatogram (Figure 3a) of purifying

HA derived from influenza B/Brisbanc/60 and SDS PAGE gel stained with Coomassie blue, loaded with fractions from the CHT ^TM Hydroxyapatite chromatography (Figure 2b)

[0040] Figure 4 depicts SDS-PAGE gel analysis and Western Blot analysis of purified HA derived from influenza A/Califomia/04/09 with high purity (>97%). The yield was about 1 1 mg_''1iter of cell culture.

[0041] Figure 5 depicts a vaccine potency assay using SRID.

DETAILED DESCRIPTION [0042] As used herein, the term "baculovirus," also known as baculoviridae, refers to a family of enveloped DNA viruses of arthropods, members of which may be used as expression vectors for producing recombinant proteins in insert cell cultures. The virion contains one or more rod-shaped nucleocapsids containing a molecule of circular sυpercoiled double-stranded DNA (Mr 54 x 10⁶- 154 x 10⁶). The virus used as a vector is generally

Autographa californica nuclear polyhedrosis virus (NVP). Expression of introduced genes is under the control of the strong promoter that normally regulates expression of the polyhedron protein component of the large nuclear inclusion in which the viruses are embedded in the infected cells.

[0043] As used herein, the term "derived from" refers to the origin or source, and may include naturally occurring, recombinant, unpurified, or purified molecules. The proteins and molecules of the present invention may be derived from influenza or non-influenza molecules.

[0044] As used herein, the term "hemagglutinin activity" refers to the ability of HA- containing proteins, VLPs. or portions thereof to bind and agglutinate red blood cells

(erythrocytes).

[0045] As used herein, the term "neuraminidase activity" refers to the enzymatic activity of

NA-containing proteins, VLPs, or portions thereof to cleave sialic acid residues from substrates including proteins such as fetuin.

[0046] As used herein, the term "heterotypic" refers to one or more different types or strains of virus.

[0047] As used herein, the term "homotypic" refers to one type or strain of virus.

[0048] As used herein, the term "macromolecular protein structure" refers to the construction or arrangement of one or more proteins.

[0049] As used herein, the term ''multivalent" vaccine refers to a vaccine against multiple types or strains of influenza virus.

[0050] As used herein, the term "non-influenza" refers to a protein or molecule that is not derived from influenza virus.

[0051] As used herein, the term "vaccine" refers to a preparation of dead or weakened pathogens, or of derived antigenic determinants, that is used to induce formation of antibodies or immunity against the pathogen. A vaccine is given to provide immunity to the disease, for example, influenza, which is caused by influenza viruses. The present invention provides vaccine compositions that are immunogenic and provide protection. In addition, die term "vaccine" also refers to a suspension or solution of an immυnogen (e.g. VLP) that is administered to a vertebrate to produce protective immunity, i.e., immunity that reduces the severity of disease associated with infection.

[0052] As used herein the Venn "substantial immunity" refers to an immune response in which when VLPs of the invention are administered to a vertebrate there is an induction of the immune system in said vertebrate which results in the prevention of influenza infection, amelioration of influenza infection or reduction of at least one symptom related to influenza virus infection in said vertebrate. Substantial immunity may also refer to a haemaggiutination inhibition (Hl) titer of ≥ 40 in a mammal wherein the VLPs of the invention have been administered and have induced an immune response.

[0053] As used herein the term "adjuvant" refers to a compound that, when used in combination with a specific immunogen (e.g. a VLP) in a formulation, augments or otherwise alters or modifies the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.

[0054] As used herein the term "immune stimulator" refers to a compound that enhances an immune response via the body's own chemical messengers (cytokines). These molecules comprise various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-I, 1L-2, IL-3, IL-4, IL- 12, IL- 13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1 ; B7.2, etc. The immune stimulator molecules can be administered in the same formulation as the influenza VLPs, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect.

[0055] As used herein an "effective dose" generally refers to that amount of the VLP of the invention sufficient to induce immunity, to prevent and/or ameliorate influenza vims infection or to reduce at least one symptom of influenza infection and/or to enhance the efficacy of another dose of a VLP. An effective dose may refer to the amount of the VLP sufficient to delay or minimize the onset of an influenza infection. An effective dose may also refer to the amount of the VLP that provides a therapeutic benefit in the treatment or management of influenza infection. Further, an effective dose is the amount with respect to the VLPs of the invention alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an influenza viral infection. An effective dose may also be the amount sufficient to enhance a subject's (e.g., a human's) own immune response against a subsequent exposure to influenza virus. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or scrum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an "effective dose" is one that prevents disease or reduces the severity of symptoms.

[0056] As used herein the term "avian influenza virus" refers to influenza viruses found chiefly in birds but that can also infect humans or other animals. In some instances, avian influenza viruses may be transmitted or spread from one human to another. An avian influenza virus that infects humans has the potential to cause an influenza pandemic, i.e., morbidity and/or mortality in humans. A pandemic occurs when a new strain of influenza virus (a virus in which human have no natural immuniiy) emerges, spreading beyond individual localities, possibly around the globe, and infecting many humans at once.

[0057] As used herein the term "seasonal influenza virus" refers to the influenza viral strains that have been determined to be passing within the human population for a given influenza season based on epidemiological surveys conducted by National Influenza Centers worldwide. These epidemiological studies, and some isolated influenza viruses, are sent to one of four World Health Organization (WHO) reference laboratories, one of which is at the Centers for Disease Control and Prevention (CDC) in Atlanta for detailed testing. These laboratories test how well antibodies made to the current vaccine react to the circulating virus and new flu viruses. This information, along with information about flu activity, is summarized and presented to an advisory committee of the U.S. Food and Drug Administration (FDA) and at a WHO meeting. These meetings result in the selection of three viruses (two subtypes of influenza A viruses and one influenza B virus) to go into flu vaccines for the following fall and winter. The selection occurs in February for the northern hemisphere and in Sq?tember for the southern hemisphere. Usually, one or two of the three virus strains in the vaccine changes each year.

[0058] As used herein the term "substantially protective antibody response" refers to an immune response mediated by antibodies against an influenza virus, which is exhibited by a vertebrate (e.g., a human), that prevents or ameliorates influenza infection or reduces at least one symptom thereof. VLPs of the invention can stimulate the production of antibodies that, for example, neutralizing antibodies that block influenza viruses from entering cells, blocks replication of said influenza virus by binding to the virus, and/or protect host cells from infection and destruction.

[0059] As used herein the term "substantially protective cellular response" refers to an immune response that is mediated by T-lymphocytes and/or other white blood cells against influenza virus, exhibited by a vertebrate (e.g., a human), that prevents or ameliorates influenza infection or reduces at least one symptom thereof. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells ("CTL"s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper -f -cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.

[0060] As used herein the term "substantial immunity in a population- wide basis" refers to immunity as a result of VLPs of the invention administered to individuals in a population. The immunity in said individual in said population results in the prevention, amelioration of influenza infection, or reduction of at least one symptom related to influenza virus infection in said individual, and prevents the spread of said influenza virus to others in the population. The term population is defined as group of individuals (e.g. schoolchildren, elderly, healthy individuals etc.) and may comprise a geographic area (e.g. specific cities, schools, neighborhoods, workplace, country, state, etc.).

[0061] As use herein, the term "antigenic formulation" or "antigenic composition" refers to a preparation which, when administered to a vertebrate, especially a bird or a mammal, will induce an immune response.

[0062] As use herein, the term "vertebrate" or "subject" or "patient" refers to any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species. Farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like are also non-limiting examples . The terms "mammals" and "animals" are included it. this definition. Both adult and newborn individuals are intended to be covered.

Purified HA Proteins

[0063] The present inventors have observed that prior art recombinant HA proteins, when administered to a host animal, fail to produce antibodies which recognize viral antigens in their native conformation (e.g. HA from the HlNl virus). In general, the failure of the prior art recombinant HA proteins can be attributed to the fact that they produced in a denatured form. The present inventors have developed an improved purification process that is less denaturing (e.g. produces HA proteins that closely resemble their native conformation). When administered to an animal, the purified HA proteins stimulate the production of antibodies that recognize viral HA antigens in their native conformation.

[0064] The purified HA proteins produced by the methods described herein have a number of diagnostic, prophylactic, and therapeutic applications. For instance, antibodies produced against the purified HA proteins are useful in potency assays such as the single radial immunodiffusion assay (SRlD). In addition, the purified HA proteins of the present invention may be administered to a subject for the generation of an antibody response, and thus find utility in the treatment and prevention of influenza virus.

[0065] In one aspect, the present invention provides purified viral proteins. In one embodiment, said purified viral proteins are recombinant proteins or chimeric proteins. In an exemplary embodiment, said purified viral protein is a recombinant or chimeric HA protein of influenza virus. In another embodiment, the HA protein is a recombinant or chimeric protein derived from an influenza virus. In some embodiments, the influenza virus is selected from the group consisting of avian influenza virus and mammalian influenza virus. In one embodiment, the mammalian influenza virus is a human influenza virus strain. In an alternative embodiment, the mammalian influenza virus is a swine influenza virus. As described herein, said HA protein may be derived from a pandemic strain or a seasonal strain of influenza virus.

[0066] In some embodiments, the HA protein is selected from the group consisting of HI , m, H3, H4, HS₅ H6, H7 H8, H9, HlO, HI 1, H 12, H 13, H 14, H 15, and H 16. In an exemplary embodiment, the HA is derived from an Hl N 1 influenza virus strain. In one embodiment, the HlNl influenza virus strain is selected from the group consisting of A/California/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007. [0067] In another embodiment, the present invention comprises purified recombinant or chimeric influenza HA with high purity. In one embodiment, the purity of said recombinant or chimeric influenza HA is at least about 60%, about 70%, about 80%, about 90%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%.

[0068] In one embodiment, the HA proteins of the present invention are found in the form of micelles (e.g. rosettes). The micelles obtainable in accordance with the invention consist of aggregates of the immunogenically active HA proteins having a rosette-like structure. The rosettes are visible in the electron microscope. The micelles are generally comprised of 20- 1 (M) HA trimers_'particle. The particle size of the micelle range from 20-40 nanometers (nm) in length. As described herein, the HA micelles of the present invention are useful for stimulating an immune response both in vitro and in vivo.

Methods of Purifying Viral Protein

[0069] In another aspect, the present invention provides a method of purifying a viral protein. In a preferred embodiment, the viral protein is an HA protein.

[0070] In one embodiment the method of purifying the HA protein comprises the steps of:

(i) Expressing said HA in a host cell;

(ii) Extracting said HA from said host cell, wherein said HA is soluble in an extraction buffer;

(iii) Purifying said HA from an extraction buffer using ion exchange chromatography, wherein a pool of fractions containing said HA is produced;

(iv) Purifying said HA from said pool in step (iii) using affinity chromatography, wherein a .second pool of fractions containing said HA is produced;

(v) Desalting said second pool in step (iv);

(vi) Purifying said HA from the desalted pool of step (v) using hydroxyapatite chromatography, wherein a third pool of fractions containing said HA is produced: and optionally

(vii) concentrating, dialyzing, and sterilizing said third pool of tractions containing HA.

[0071] In some embodiments, the HA protein is selected from the group consisting of Hl , H2, H3, H4, H5, H6, H,7 H8, H9, H 10, H 11 H12, H 13, H 14 , H I S and H 16. In an exemplary embodiment, the HA is derived from an Hl Nl influenza virus strain. In one embodiment, the H1N1 influenza virus strain is selected from the group consisting of A/CaKfornia/04/09, A/New Caledonia/20/1999, A/Solomon ls/3/200ό, and

A''Brisbane/59/2007.

[0072] In another embodiment, the fractions elυted from columns in steps (iii), (Iv), (vi) are analyzed with SDS-PAGE gel stained by Coomassie blue and Western Blot by antibody against said viral protein, respectively, to determine fractions contain majority of said viral protein, wherein said fractions are subsequently combined together to form a pool. To reach high purity, yield of said purified viral protein can be sacrificed in a reasonable range.

[0073] In another embodiment, the purity of finally purified viral protein is at least about 60%, about 70%, about 80%, about 90%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%. In an exemplary embodiment, the purity of said viral protein is at least about 95%.

Ion Exchange Chromato graph y

[0074] Ion Exchange Chromatography relies on charge-charge interactions between the proteins in your sample and the charges immobilized on the resin of your choice. Ion exchange chromatography can be subdivided into cation exchange chromatography, in which positively charged ions bind to a negatively charged resin; and anion exchange chromatography, in which the binding ions are negative, and the immobilized functional group is positive. Once the solutes are bound, the column is washed to equilibrate it in your starting buffer, which should be of low ionic strength, then the bound molecules are eluted off using a gradient of a second buffer which steadily increases the ionic strength of the elute solution. Alternatively, the pH of the elute buffer can be modified as to give your protein or the matrix a charge at which they will not interact and your molecule of interest elutes from the resin. If one knows the pH used during running and need to decide what type of ion exchange to use, protein sequence can be inputted into software to predict the charge status of the protein. If it is negatively charged at the pH, use an anion exchanger; if it is positive, use a cation exchanger. In many cases it may be more advantageous to actually select conditions at which the protein will flow through while the contaminants will bind. This mode of binding is often referred to as "flow through mode". Tins is a particularly good mode to use in the case of anion exchange. Here one could use this type of mode to bind up endotoxins or other highly negatively charged substances well at the same time relatively simply flowing the protein through the matrix. Non-limiting examples of ion exchanger resins used for packing columns are, Q-resin, Di Ethyl AminoEthane (DEAE) resin, dimethylaminoethyl (DMAE) resin, Trimethylaminoethyl (TMAE) resin, SOf resin, COO ~ resin, and those described in U.S. Patent Nos. 7,279,244, 7,097,771, 7,037,949, 7,022,743, 6,989,212, 6,977,314, 6,972,091, 6,488,859, 6,486,222, 6,437,010, 6,422,492, 6,265,224, 6,241 ,980, 6,232,353, 6,203,708, 6,193,962, 6,077,532, 6,060,526, 5,962,183, 5,922,171 , 5,858,139, 5,808,042, 5,759,942, 5,736,052, 5,648,400, 5,637,627, 5,622,997, 5,614,352, 5,545,798, 5,451,309, 5,441,991, 5,428,075, 5,403,492, 5,397,477, 5,374,756, 5,368,818, 5,352,345, 5,279,744, 5,278,193, 5,275,820, 5,248,435, 5,071,646, 5,055,694, 5,004,71 1, 4,944,878, 4,917,806, 4,891,138, 4,847,006, 4,788,223, 4,780,239, 4,770,783, 4,732,705, 4,719,242, 4,719,241 , 4,700,723, 4,698,153, 4,680,170, 4,671,898, 4,666,856, 4,628,837, 4,608,279, 4,576,969, 4,569,765, 4,559,170, 4,554,378, 4,553,034, 4,533,678, 4,524,153, 4,495,152, 4,453,99.1 , 4,451,581 , 4,451,440, 4,446,252, 4,430,445, 4,387,075, 4,366,261, 4,314,905, 4,298,578, 4,245,053, 4,216,073, 4,192,921, 4,184,019, 4,177,331, 4,135,880, 4,1 19,580, 4,116,546, 4,1 1 1 ,856, 4,104,204, 4,065,388, 4,045,379, 4,008,171 , 3,998,627, 3,995,009, and 3,943,074.

[0075] In one embodiment of the present invention, said ion exchange chromatography in step (iii) is an anion exchange chromatography (AEC), wherein the surface charge of the solutes (proteins, nucleic acids, endotoxin) which bind will be net negative, thus to get binding of a specific protein one should be above the pi of that protein. Common anion exchange resins used to pack anion exchange columns are Q-resin (a Quaternary amine), DEAE resin (DiEthylAminoEthane), dimethylamraoethyl (DMAE) resin, Trimethylaminoethyl (TMAB) resin, SCV resin, and COO ^" resin. Anion exchange chromatography is often used as a primary chromatography step due to its high capacity, (Matrices can bind from 10 to 100 mg of protein per ml) and ability to bind up and separate fragmented nucleic acids and lipopolysaccharides from the initial slurry. Typically, anion exchange chromatography is performed using buffers at a pH between 7 and 10 and running a gradient from a solution containing just this buffer to a solution containing this buffer with NaCl. The salt in the solution competes for binding to the immobilized matrix and releases the protein from its bound state at a given concentration. Proteins separate because the amount of salt needed to compete varies with the external charge of the protein. Uses of anion exchange chromatography include initial clean up of a crude slurry, separation of proteins from each other, concentrating a protein, and the removal of negatively charged endotoxin from protein preparations.

[0076] Thus, in one embodiment of the present invention, the ion exchange chromatography in said step (iii) uses a anion exchange column packed with Trimethylaminoethyl (TMAE) anion exchanger resins (e.g., Fractoprep® EMD TMAE resins).

Affinity Chromatography

[0077] Affinity chromatography is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Affinity chromatography combines the size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules. Due to its interdisciplinary' nature, affinity chromatography has been the means by which many scientists from different disciplines have been introduced to the exciting fields of modem biology. Affinity chromatography can be used to purify and concentrate a molecule from a mixture into a buffering solution, reduce the amount of a molecule in a mixture, discern what biological compounds bind to a particular molecule, such as drugs, and purify and concentrate an enzyme solution. To perform affinity chromatography, usually the starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate, growth medium or blood serum. The molecule of interest wϋl have a well known and defined property which can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in solution will not become trapped as they do not possess this property. The solid medium can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as elution. Binding to the solid phase may be achieved by column chromatography, whereby the solid medium is packed onto a chromatography column, the initial mixture run through the column to allow binding, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure (as opposed to HPLC or FPLC). Alternatively binding may be achieved using a batch treatment, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase (by eentrirugation for example), removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the eluate. Sometimes a hybrid method is employed, the binding is done by the batch method, then the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column. A third method, expanded bed adsorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase. Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and antibody purification from blood serum. The most common use of affinity chromatography is for the purification of recombinant proteins. Proteins with a known affinity are tagged in order to aid their purification. The protein may have been genetically modified so as to allow it to be selected for affinity binding, this is known as a fusion protein. Non-limiting exemplary tags include His-tags and GST (glutathione-S-transferase) tags.

[0078] Non-limiting examples of affinity chromatography are, antibody affinity chromatography, immobilized metal ion affinity chromatography, and lectin affinity chromatography. Thus, in one embodiment of the present invention, said affinity chromatography in step (iv) in the purification method mentioned above is lectin affinity chromatography {e.g., Lentil lectin Sepharose 4B chromatography, GH).

Desalting.Chrom.atography and Dialysis

[0079] Solution containing soluble protein needs to be desalted before it is loaded on certain type of column (e.g., hydroxyapatite column). Non-limiting exemplary desalting methods are, desalting column, desalting tips, and dialysis.

[0080] In one embodiment of the purification method step (v) of the present invention mentioned above, said pool containing viral protein is desalted it by passing through a desalting column, a desalting tip, or by dialysis. In one embodiment of the present invention, a desalting column can be selected from the group consisting of Pierce desalting column, Httrap™ Desalting Column (GE Health), HiPrep™ Desalting Column (GE Health), PD-10 Desalting column (GE Health), and Uptima desalting column. In a further embodiment, said desalting column is packed with Sephadex™ G-25.

[0081] Dialysis is one type of diffusion, or osmosis. It allows the passage of small molecules such as salt but not larger ones such as protein to pass through a semi or partially permeable membrane bag or tubing made from regenerated cellulose or cellophane. It can be used in clinical circumstances to ensure a filtered flow of molecules, preventing the flow of larger solute molecules, or used in purification field to desalt. Small molecules such as salt can be washed out of a solution which is contained in a bag or pumped through the tubing into a solvent, usually water, which surrounds the bag or tubing and in which small molecules can be flushed away. A solution containing several types of molecules, usually glucose and starch, is placed into a semi-permeable dialysis bag, such as a cellulose membrane with pores, and the bag is sealed. The sealed dialysis bag is placed in a container of a different exchange solution, or water. Molecules small enough to pass through the tubing (water, salts, monosaccharides, and other small molecules) tend to move into or out of the dialysis bag in the direction of decreasing concentration, therefore displaying diffusion. Larger molecules (such as proteins, or polysaccharides) that have dimensions significantly greater than the pore diameter are retained inside the dialysis bag. A hypotonic solution crosses the semipermeable membrane into the hypertonic solution in an attempt to reach equilibrium. The exchange solution in the container can be replaced for several times to reach desired desalting goal.

[0082] A semi or partial permeable membrane, also termed selectively permeable membrane, or differentially permeable membrane, is a membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally specialized facilitated diffusion. The rate of passage depends on the pressure, concentration, and temperature of the molecules or solutes on either side, as well as the permeability of the membrane to each solute. Depending on the membrane and the solute, permeability may depend on solute size, solubility, properties, or chemistry. Non-limiting exemplar}' semi-permeable membranes are, thin film composite membranes (TFC or TFM), cellulose ester membrane (CEM), charge mosaic membrane (CMM), bipolar membrane (BPM), anion exchange membrane (AEM), alkali anion exchange membrane (AAEM) and proton exchange membrane (PEM). Selectively permeable membrane can be classified by a perimeter called molecular cut-off (MWCO), which refers to the molecular weight of the biggest molecule that can pass through the membrane.

[0083] Thus in one embodiment of the purification method of the present invention, dialysis bag, tubing, cassette or a stirred cell with selectively permeable membrane can be used to desalt a pool containing viral protein to be purified in step (v) mentioned above. In another embodiment, said dialysis bag, tubing, cassette, or stilted cell comprises a selective permeable membrane having a MVVCO of about 5 kD, about 1OkD, about 15 kD, about 20 kD_> about 25 kD, about 30 kD, about 35kD, about 35kD, or about 4OkD. For example, the bag/tubing comprises a selective permeable membrane having a MWCO of about 20 to about 30 kD (e.g., slide-A-lyzer dialysis cassette or Ami con Stir Cells).

Hvdroxyapatite Chromatography [0084] Hydroxyapatite, also called hydroxyapatite, is a naturally occurring mineral form of calcium apatite with the formula Ca_S(POt)₃(OH) (a.k.a. Ca₁₀(P(M)₆(OH)₂). Hydroxyapatite is the hydroxyl end member of the complex apatite group. The OH- ion can be replaced by fluoride, chloride or carbonate. It crystallizes in the hexagonal crystal system.

[0085] Hydroxyapatite can be used in chromatography for purification. The mechanism of hydroxyapatite chromatography is complicated and has been described as "mixed-mode" ion exchange. It involves nonspecific interactions between positively charged calcium ions and negatively charged phosphate ions on the stationary phase hydroxyapatite resin with protein negatively charged carboxyl groups and positively charged amino groups. It may be difficult to predict the effectiveness of hydroxyapatite chromatography based on physical and chemical properties of the desired protein to be purified. For elution, a buffer with increasing phosphate concentration is typically used. Non-limiting exemplary hydroxyapatite that can be used to pack column for chromatography are, natural hydroxyapatite and synthetic hydroxyapatite (e.g., crystalline hydroxyapatite, ceramic hydroxyapatite).

[0086] Thus in one embodiment of the purification method of the present invention, solution containing desired viral protein can be further purified in step (iv) mentioned above by a hydroxyapatite column, wherein said hydroxyapatite column is packed with natural hydroxyapatite or synthetic hydroxyapatite, wherein the synthetic hydroxyapatite is crystalline hydroxyapatite or ceramic hydroxyapatite. For example, the column is packed with spherical, macroporous form of CHT™ hydroxyapatite, Type I (ceramic hydroxyapatite).

[0087] The present invention further comprises method of concentrating, dialyzing and sterilizing viral protein product purified in above mentioned purification method. In one embodiment, the viral protein product is further concentrated and dialyzed using a stirred cell with selectively permeable membrane as described above. For example, the viral protein product is concentrated and dialyzed using a Millipore Stirred Cell with selectively permeable membrane against DPBS buffer. In one embodiment, the selective membrane has a MWCO is about 30 KD. A minimum of three additions of 10-fold buffer volume is needed for dialysis. Sterile Filtration is performed using a micron filter (e.g., a 0.2 micron SFCA (or PVDF) filter).

VLPs and Methods of making VLPs

[0088] In general, virus-like particles (VLPs) lack a viral genome and, therefore, are noninfectious. In addition, virus-like particles can often be produced by heterologous expression and can be easily purified. Most VLPs comprise at least a viral core protein. This core protein usually drive* budding and release of particles from a host cell. Examples of such proteins comprise RSV M, influenza Ml, HIV gag and vesicular stomatis virus (VSV) M protein. In general, VLPs are useful for preparing antigenic formulation and/or vaccines against infectious agents, e.g. influenza.

|OO895 There are 16 different hemagglutinin (HA) and 9 different neuraminidase (NA) all of which have been found among wild birds. Wild birds are the primary natural reservoir for all types of influenza A viruses and are thought to be the source of all types of influenza A viruses in all other vertebrates. These subtypes differ because of changes in the hemagglutinin (HA) and neuraminidase (NA) on their surface. Many different combinations of HA and NA proteins are possible. Each combination represents a different type of influenza A virus. In addition, each type can be further classified into strains based on different mutations found in each of its 8 genes. The present invention describes the production of influenza vaccine candidates or reagents comprised of influenza proteins that self-assemble into functional VLPs. AU combinations of viral proteins must be co-expressed with a matrix protein 1 (Ml).

[0090] The invention described herein provides VLPs comprising a purified influenza HA protein. In one embodiment, the purity of the HA protein is at least about 95%. In another embodiment, the HA protein is a recombinant or chimeric protein derived from an influenza virus. In some embodiments, the influenza virus is selected from the group consisting of avian influenza virus and mammalian influenza virus. In one embodiment, the mammalian influenza virus is a human influenza virus strain. In an alternative embodiment, the mammalian influenza virus is a swine influenza virus. As described herein, said HA protein may be derived from a pandemic strain or a seasonal strain of influenza virus.

[0091] In some embodiments, the HA protein is selected from the group consisting of Hl, H2, H3, H4, HS, 116, H7 H8, H9, HlO, HI l, H 12, H 13, H 14, H 15, and H 16. In an exemplary embodiment, the HA is derived from an HlN 1 influenza virus strain. Jn one embodiment, the HlNl influenza virus strain is selected from the group consisting of A/California/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.

[0092] In some embodiments, the VLPs further comprises at least one influenza matrix protein (Ml). In one embodiment, the Ml protein is derived from an H lNl influenza virus strain. In one embodiment, the HlNl influenza virus strain is selected from the group consisting of A/Califomia/04/09, A/New Caledonia/20/1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007. In some alternative embodiments, the Ml protein is derived from an avian influenza virus strain.

[0093] In one embodiment, the VLPs further comprise an influenza NA protein. In another embodiment, said NA is derived from an avian or mammalian influenza virus, and is selected from the group consisting of Nl , N2, N3, N4, N5, N6, N7, N8 and N9. In one embodiment, the NA protein is derived from an HlNl influenza virus strain. In one embodiment, the H lNl influenza virus strain is selected from the group consisting of A/California/04/09, A/New CaIedonia/20/1999, A/Solomon ls/3/2006, and A/Brisbane/59/2007.

[0094] In another embodiment, the invention comprises VLPs that consist essentially of HA, NA and Ml. Said HA and NA can be from the above list of HA and NA. These VLPs may comprise additional influenza proteins and/or protein contaminates in negligible concentrations. These VLPs contain HA, NA and Ml and may contain additional cellular constituents such as cellular proteins, baculovirus proteins, lipids, carbohydrates etc., but do not contain additional influenza proteins (other than fragments of Ml, HA and/or NA). In another embodiment, the HA and/or the NA may exhibit hemagglutinin activity and/or neuraminidase activity, respectively, when expressed on the surface of VLPs.

[0095] The invention also encompasses variants of the said influenza proteins expressed on or in the VLPs of the invention. The variants may contain alterations in the amino acid sequences of the constituent proteins. The term "variant" with respect to a polypeptide refers to an amino acid sequence that is altered by one or more amino acids wilh respect to a reference sequence. The variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. Alternatively, a variant can have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the ait, for example, DNASTAR software.

[0096] Natural variants can occur due to antigenic drifts. Antigenic drifts are small changes in the viral proteins that happen continually over time. Thus, a person infected with a particular flu virus strain develops antibody against that virus, as newer virus strains appear, the antibodies against the older strains no longer recognize the newer virus and reinfection can occur. This is why there is a new vaccine for influenza each season. In addition, some changes in an influenza virus can cause influenza virus to cross species. For example, some avian influenza viruses developed genetic variations associated with the capability of crossing the species barrier. Such a virus is capable of causing a pandemic because people have no natural immunity to the virus and the virus can easily spread from person to person. These naturally occurring variations of the influenza proteins are an embodiment of the invention.

[0097] General texts which describe molecular biological techniques, which are applicable to the present invention, such as cloning, mutation, cell culture and the like, include Berger and Khtimel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning--A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et ai , eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., ("Ausubel"). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the cloning and mutation of HA and/or NA molecules, etc. Thus, the invention also encompasses using known methods of protein engineering and recombinant DNA technology to improve or alter the characteristics of the influenza proteins expressed on or in the VLPs of the invention. Various types of mutagenesis can be used to produce and/or isolate variant HA, NA and/or Ml molecules and/or to further modify/mutate the polypeptides of the invention. They include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction- purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. Mutagenesis, e.g., involving chimeric constructs, is also included in the present invention. In one embodiment, mutagenesis can be guided by known information of the naturally occurring molecule or altered or mutated naturally occurring molecule, e.g., sequence, sequence comparisons, physical properties, crystal structure or the like.

[0098] Methods of cloning influenza proteins are known in the art. For example, the influenza gene encoding a specific influenza protein can be isolated by RT-PCR from polyadenylated mRNA extracted from cells which had been infected with an influenza virus. The resulting product gene can be cloned as a DNA insert into a vector. The term "vector" refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, pro~vϊru9es, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating. In many, but not all, common embodiments, the vectors of the present invention are plasmids or bacmids.

[0099] The invention also provides for methods of producing VLPs, said methods comprising expressing an avian, pandemic and/or seasonal influenza proteins under conditions that allow VLP formation. Depending on the expression system and host cell selected, the VLPs are produced by growing host cells transformed by an expression vector under conditions whereby the recombinant proteins are expressed and VLPs are formed. The selection of the appropriate growth conditions is within the skill or a person with skill of one of ordinary skill in the art.

[00100] In one embodiment, VLPs can expressed in eukaryotic cells and/or prokaryotic cells. Among eukaryotic host cells are yeast, insect, avian, plant. C. elegam (or nematode), and mammalian host cells. Non limiting examples of insect cells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21, Trichoplusia ni cells, e.g. High Five cells, and Drosophila S2 cells. Examples of fungi (including yeast) host cells are S. cerevisiae, Kluyreromyces lactis (K. laclis), species of Candida including C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharornyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (MEK) cells, and African green monkey cells. CVI cells, HeLa cells, MDCK cells, Vero, and Hep-2 cells. Xenopus laevis oocytes, or other cells of amphibian origin_^ may also be used. Prokaryotic host cells include bacterial cells, for example, E. coli, B. suhtilis, and mycobacteria. A preferred host cell is a SrP insect cell.

[00101] Methods to grow cells engineered to produce VLPs of the invention include, but are not limited to, batch, batch-fed, continuous and perfusion cell culture techniques. Cell culture means the growth and propagation of cells in a bioreactor (a feπnentation chamber) where cells propagate and express protein (e.g. recombinant proteins) for purification and isolation. Typically, cell culture is performed under sterile, controlled ternperaiure and atmospheric conditions in a bioreactor. A bioreactor is a chamber used to culture cells in which environmental conditions such as temperature, atmosphere, agitation and/or pH can be monitored. In one embodiment, said bioreactor is a stainless steel chamber. In another embodiment, said bioreactor is a pre-sterilized plastic bag (e.g. Cellbag®, Wave Biotech, Bridgewater, NJ). In other embodiment, said pre-sterilized plastic bags are about 50 L to 1000 L bags.

[00102] The VLPs are then isolated using methods that preserve the integrity thereof, such as by gradient centrifύgation, e.g., cesium chloride, sucrose and iodixanol, as well as standard purification techniques including, e.g., ion exchange and gel filtration chromatography.

[00103] The following is an example of how VLPs of the invention can be made, isolated and purified. Usually VLPs are produced from recombinant cell lines engineered to create a VLP when said cells are grown in cell culture (see above). Production of VLPs may be accomplished as illustrated in Examples. A person of skill in the art would understand that there are additional methods that can be utilized to make and purify VLPs of the invention, thus the invention is not limited to the method described.

[00104] Production of VLPs of the invention can start by seeding Sf9 cells (non-infected) into shaker flasks, allowing the cells to expand and scaling up as the cells grow and multiply (for example from a 125-ml flask to a 50 L Wave bag). The medium used to grow the cell is formulated for the appropriate cell line (preferably serum free media, e.g. insect medium ExCell-420, JRH). Next, said cells are infected with recombinant baculovirus at the most efficient multiplicity of infection (e.g. from about 1 to about 3 plaque forming units per cell). Once infection has occurred, the influenza HA, NA and Ml proteins are expressed from the virus genome, self assemble into VLPs and are secreted from the cells approximately 24 to 72 hours post infection. Usually, infection is most efficient when the cells are in mid-log phase of growth (4-8 * IO⁶ cells/ml) and are at least about 90% viable.

[00105] The present invention also comprises methods of purifying VLPs, wherein the purified VLPs have high purity and high potency as vaccine. Said methods of purifying VLPs comprises steps consisting of:

a. concentrating clarifying culture medium containing VLP; and

b. purifying concentrated diafiltered VLPs on a discontinuous sucrose gradient; and c. further purifying VLPs by anion exchange chromatography, or isopycnic sucrose cushion centrifugation.

[00106] In one embodiment, the anion exchange chromatography in step b is a TMAE anion exchange chromatography

[00107] For example, VLPs of trie invention can be harvested approximately 48 to 96 hours post infection, when the levels of VLPs in the cell culture medium are near the maximum but before extensive cell lysis. The Sf9 cell density and viability at the time of harvest can be about 0.5x H)⁶ cells/ml to about 1.5 x K)⁶ cell.s/ml with at least 20% viability, as shown by dye exclusion assay. Next, the medium is removed and clarified. NaCl can be added to the medium to a concentration of about 0.4 to about 1.0 M, preferably to about 0.5 M, to avoid VLP aggregation. The removal of cell and cellular debris from the cell culture medium containing VLPs of the invention can be accomplished by tangential flow filtration (TFF) with a single use, pre-sterilized hollow fiber 0.5 or 1.00 μrn filter cartridge or a similar device.

[00108] Next, VLPs in the clarified culture medium can be concentrated by ultrafiltration using a disposable, pre-sterilized 500,000 molecular weight cut off hollow fiber cartridge. The concentrated VLPs can be diafiltrated against 10 volumes pH 7.0 to 8.0 phosphate- buffered saline (PBS) containing 0.5 M NaCl to remove residual medium components.

[00109] The concentrated, diafiltered VLPs can be furthered purified on a 20% to 60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaCl by centrifugarion at 6,500 x g for 18 hours at about 4° C to about 10° C. Usually VLPs will form a distinctive visible band between about 30% to about 40% sucrose or at the interface (in a 20% and 60% step gradient) that can be collected from the gradient and stored. This product can be diluted to comprise 200 mM of NaCl in preparation for the next step in the purification process. This product contains VLPs and may contain intact baculovirus particles,

[00110] Further purification of VLPs can be achieved by anion exchange chromatography, or 44% isopycnic sucrose cushion centrifugation. In anion exchange chromatography, the sample from the sucrose gradient (see above) is loaded into column containing a medium with an anion (e.g. Matrix Fractogel EMD TMAE) and eluded via a salt gradient (from about 0.2 M to about 1.0 M of NaCl) that can separate the VLP from other contaminates (e.g. baculovirus and DNA/R.NA). fn the sucrose cushion method, the sample comprising the VLPs is added to a 44% sucrose cushion and centrifυged for about 18 hours at 30,000 g. VLPs form a band at the top of 44% sucrose, while baculovirus precipitates at the bottom and other contaminating proteins stay in the 0% sucrose layer at the top. The VLP peak or band is collected.

[00111] The intact baculovirus can be inactivated, if desired. Inactivation can be accomplished by chemical methods, for example, formalin or β-propyl lactone (BPL). Removal and/or inactivation of intact baculovirus can also be largely accomplished by using selective precipitation and chromatographic methods known in the art, as exemplified above. Methods of inactivation comprise incubating the sample containing the VLPs in 0.2% of BPL for 3 hours at about 25 °C to about 27 °C. The baculovirus can also be inactivated by incubating the sample containing the VLPs at 0.05% BPL at 4 °C for 3 days, then at 37 °C for one hour.

[00112] After the itiactivation/removal step, the product comprising VLPs can be run through another diafϊltration step to remove any reagent from the inactivation step and/or any residual sucrose, and to place the VLPs into the desired buffer (e.g. PBS). The solution comprising VLPs can be sterilized by methods known in the art (e.g. sterile filtration) and stored in the refrigerator or freezer.

[00113] The above tecliniques can be practiced across a variety of scales. For example, T- flasks, shake-flasks, spinner bottles, up to industrial sized bioreactors. The bioreactors can comprise either a stainless steel tank or a pre-sterilized plastic bag (for example, the system sold by Wave Biotech, Bridgewater, NJ). A person with skill in the art will know what is most desirable for their purposes.

[00114] In another embodiment of the invention, said VLPs comprise more than one protein from an infectious agent. Tn this embodiment, said VLPs are multivariant VLPs capable of inducing an immune response to several proteins from infectious agents. In one embodiment said VLPs comprise proteins from at least two different influenza viruses. For example said multivariant VLPs can comprise a HA and/or NA from a seasonal influenza virus A and/or B and/or from an avian influenza virus. This embodiment also comprises the presentation of HA and/or NA of the three influenza viruses (two subtypes of influenza A viruses and one influenza B virus) that are chosen by WHO and the CDC to be in the flu vaccines for the fall and winter in a single VLP. In another embodiment, said multivariant VLPs comprise proteins from several viruses, bacteria and/or parasites. For example, said VLPs comprise proteins from influenza and RSV, influenza, RSV and parainfluenza.

[00115] In another embodiment, said chimeric proteins comprise a fusion between the influenza HA with the protein, or a portion thereof, from an infectious agent. In another embodiment, said chimeric proteins comprise a fusion between the proteins, or a portion thereof, of two infectious agents or antigenic variations of the same agent. Said fusion protein will comprise antigenic agents from each protein from said infectious agent. In another embodiment, said chimeric protein comprises an amino acid linker between the proteins. An example of this embodiment is a fusion between the influenza HA and the RSV F protein. [00116] A protein that may be expressed on the surface of chimeric VLPs of the invention can be derived from viruses, bacteria, rungi and/or parasites. In other embodiments, the proteins expressed on the surface of said VLPs may be tumor or cancer antigens. The proteins derived from viruses, bacteria, fungi and/or parasites can induce an immune response (cellular and/or humoral) in a vertebrate that which will prevent, treat, manage and/or ameliorate an infectious disease in said vertebrate.

[00117] Non-limiting examples of viruses from which said infectious agent proteins can be derived from are the following: Orthopoxvirus (e.g., Cowpoxvirus, Monkeypox virus, Vaccinia virus. Variola virus, Parapoxvirus, Bovine papular stomatitis vims, Orf virus, Pseudocowpox virus, MoHuscipoxvims, Molluscum contagiosuin virus, Yatapoxvirus, Tanapox virus, Yaba monkey tumor virus), Simplexvirus (e.g., Human herpesvirus 1 (Herpes simplex virus 1), Human herpesvirus 2 (Herpes simplex virus 2), Human herpesvirus 3 (Varicella-zoster vims)), Cytomegalovirus (e.g._* Human herpesvirus 5 (Human cytomegalovirus)), Roseolvirus (e.g., Human herpesvirus 6_> Human herpesvirus 7), Lymphocryptovirus (e.g., Huam herpesvirus 4), Rhadinovirus (e.g., Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus)), Mastadenovirus (e.g., Human adenovirus A, Human adenovirus B, Human adenovirus C. Human adenovirus D, Human adenovirus E, Human adenovirus F), Polyomavirus (e.g. BK polyomavirus. Human polyomavirus, JC polyomavirus), Alphapapillomavirus (e.g., Human papillomavirus 2, Human papillomavirus 10, Human papillomavirus 6, Human papillomavirus 7, Human papillomavirus 16, Human papillomavirus 18, Human papillomavirus 26, Human papillomavirus 32, Human papillomavirus 34, Human papillomavirus 53, Human papillomavirus 54, Human papillomavirus 61, Human papillomavirus 71 , Human papillomavirus cand90), Betapapillomavirυs (e.g. Human papillomavirus 5, Human papillomavirus 9, Human papillomavirus 49, Human papillomavirus cand92, Human papillomavirus cand96). Gammapapillomavirus (e.g., Human papillomavirus 4, Human papillomavirus 48, Human papillomavirus 50, Human papillomavirus 60, Human papillomavirus 88), Mupapillomavirus (e.g.. Human papillomavirus 1 , Human papillomavirus 63 ),Erythro virus (e.g., BI 9 virus), Orthohepadnavirus (e.g., Hepatitis B virus), Deltaretrovirus (e.g., Primate T-lymphotropic virus 1, Primate T-lymphotropic virus 2), Lentivirus (e.g., Human immunodeficiency vims 1 , Human immunodeficiency virus 2), Orthoreovirus (e.g., Mammalian orthoreυvirus), Orbivirus (e.g.. African horse sickness virus, Changuinola virus, Corriparta virus, Orungo virus). Rotavirus (e.g., Rotavirus A, Rotavirus B), Marburgvirus (e.g., Lake Victoria marburgvirus), Ebolvirus (e.g.. Ivory Coast ebolavirus, Restoπ ebolavirus, Sudan ebolavirus, Zaire ebolavirus), Respirovirus (e.g., Human parainfluenza virus 1 , Human parainfluenza virus 3), Morbillivirus (e.g., Measles virus (Edmonston virus)), Rubulavirus, (e.g., Human parainfluenza virus 2, Human parainfluenza virus 4, Mumps virus), Henipavirus (e.g.,

Hendravirus, Nipahvirus), Pneumovirus (e.g., Human respiratory syncytial virus),

Metapneumovirus (e.g., Human metapneurnovirus), Vesiculovirus (e.g., Chandipura virus,

Cocal virus, Isfahan virus, Piry virus, Vesicular stomatitis Alagoas virus, Vesicular stomatitis

Indiana virus, Vesicular stomatitis New Jersey virus, Lyssavirus, Australian bat lyssavtrus,

Rabies virus), Influenzavirus A (e.g., Influenza A virus), Influenzavirus B (e.g., Influenza B virus), Influenzavirus C (e.g., Influenza C virus), Bunyavirus (e.g., Bunyamwera virus,

Bwamba virus, California encephalitis virus, Guama virus, Oriboca virus, Oropouche virus,

Hantavims, Andes virus, Hantaan virus, Puumala virus, Seoul virus, Dobrava-Belgrade vims,

Bayou virus, Black Creek Canal virus, New York vims, Sin Notnbre virus), Nairovirus (e.g.,

Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus), Phlebovirus (e.g., Rift

Valley fever virus, Sandfly fever Naples virus), Arenavirus (e.g., JLassa virus, Lymphocytic choriomeningitis virus, Guanarito vims, Junin virus, Machupo virus, Sabia virus), Deltavirus

(e.g., Hepatitis delta virus), Coronavirus (e.g., Human coronavirus 229E, Human coronavirus

OC43, Human enteric coronavirus, Severe acute respiratory syndrom coronavirus),

Torovims, Enterovirus (e.g., Human enterovirus A, Human enterovirus B, Human enterovirus

C, Human enterovirus D, Poliovirus), Rhinovirus (e.g., Human rhinovirus A, Human rhinovirus B), Hepatovims (e.g., Hepatitis A virus), Parechovirus (e.g., Human parechovirus), Norwalk virus (e.g., Sapovirus), Sapporo virus, Hepatitis E virus,

Mamastrovirus (e.g., Human astrovims), Alphavims (e.g., Chikungunya virus, O'nyong- nyong virus, Mayaro virus, Ross River vims, Barmah Forest virus, Sindbis vims, Ockelbo virus, Venezuelan equine encephalitis vims, Western equine encephalitis virus, Eastern equine encephalitis vims), Rubivirus (e.g., Rubella virus), Flavivirus (e.g., Kyasanur Forest disease vims, Omsk hemorrhagic fever virus, Powassan virus, I^uping ill virus, Tick-borne encephalitis virus, Dengue virus, Japanese encephalitis vims, Murray Valley encephalitis virus, St. Louis encephalitis virus, West Nile vims, l^'lheus virus, Yellow fever virus), Apoi virus, Hepatitis C vims, GB virus B, GB vims A, Prions (e.g., Creutzfeldt-Jakob-Disease,

Kuru, Gerstmann-Straussler-Scheinker syndrome, Fatal familial insomnia).

[00118] Non-limiting examples of bacteria from which said infectious agent proteins can be derived from are the following: B. pertussis, Leptospira pomona, S. paratyphi A and B, C. diphtheriae, C. tetani, C. botulimim, C, perfnngens, C. feseri and other gas gangrene bacteria, β. anthracis, P, pestis, P. multocida, Neisseria meningitidis, N. gonorrhea?., Hemophilus influenzae, Actinomyces {e.g., Norcardia), Acinetobacter, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e .g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Campylobacter, Chlamydia, Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), E. coli (e.g., Enterotoxigenic B. coli and Enterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter aerogems), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Serratia, Yersinia, Shigella), ErysipeJothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis). Vibrio (e.g., Vibrio cholerae), Pasteυrellacea, Proteus, Pseudomonas (e.g., Psetidomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Meningococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), Ureaplasmas. Treponema pollidum, Staphylococcus aureus, Pasteurella haemolytica, Corynelxicterium diptheriae toxoid, Meningococcal polysaccharide, Bordelella perlusis, Streptococcus pneumoniae, Clostridium telani toxoid, and Mycobacterium bovis.

[001 j Non-limiting examples of parasites from which said infectious agent proteins can be derived from are the following: leishmaniasis (teishmania tropica mexicana, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, iΛiishmania chagasi), trypanosomiasis (Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense), toxoplasmosis (Toxoplasma gondii) , schistosomiasis (Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Schistosoma mekongi, Schistosoma inter calatum), malaria (Plasmodium virax, Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale) Amebiasis (Entamoeba histolytica), Babesiosis (Babesiosis microti), Cryptosporidiosis (Cryptosporidium parvum), Dientamoebiasis (Dientamoeba fragilis), Giardiasis (Giardia lamblia), Helminthiasis and Trichomonas (Trichomonas vaginalis).

[002] Non-limiting examples of fungi from which said glycoproteins can be derived are from the following: Absidia (e.g. Absidia corymbifera), Ajcllomyces (e.g. Ajellomyces capsulatus, Ajellomyces dermatilidis), Arthroderma (e.g. Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gyjxseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii), Aspergillus (e.g. Aspergillus βtmigatus, Aspergillus niger), Candida (e.g. Candida albicans, Candida albicans var. stellatoidea, Candida dublinensis, Candida glabrata, Candida guilliermondii (Pichia guilliermondii), Candida lcrusei (Issatschenkia orientalis), Candida parapsilosis, Candida pelliculosa (Pichia anomala), Candida tropicalis, Coccidioides {e.g. Coccidiυides immitis), Cryptococcus (e.g. Cryptococcus neoformans (Filobasidiella neoformans), Histoplasma (e.g. Histoplasma capsulatum (Ajellomyces capsidalus), Microsporum (e.g. Microsporum canls (Λrthroderma otae), Microsporum fttlvurn (Arthroderma fulvum), Microsporum gypseum, Genus Pichia (e.g. Pichia anomala, Pichia guilliermondiϊ), Pneumocystis (e.g. Pneumocystis jirovecii), Cryptosporidium, Malassezia furfur, Paracoccidiodes.

[00119] The above lists are meant to be illustrative and by no means axe meant to limit the invention to those particular bacterial, viral or parasitic organisms.

Vaccine Potency Assay

[00120] The present invention provides purified viral protein and/or purified VLPs that can be used as a standard in a vaccine potency assay. In one embodiment, said viral protein is a recombinant viral protein or a chimeric viral protein. In a further embodiment, said purified recombinant viral protein is a recombinant or chimeric HA. In another embodiment, said recombinant or chimeric HA is selected from the group consisting of Hl, H2, H3, H4, H5, Hό, 11,7 H8, H9, H10, HI l, H 12, H 13, H14 , H15 and H 16. In another embodiment, said VLPs comprises or essentially comprises an influenza HA, an matrix protein, and optionally an influenza NA. In another embodiment, said purified recombinant or chimeric HA has a purity of at least about 95%. In another embodiment, said purified recombinant or chimeric HA and/or said purified VLl³S can be used in a potency assay of a vaccine produced by industry, governmental institutions (e.g., N1H), no-profit organizations (e.g., WHO) or regulatory bodies (e.g., FDA).

[00121] Commonly used vaccine potency assays include, but are not limited to, direct challenge of vaccinated subject with infectious virus (e.g., ED₅₀ (vaccine dosage capable of protecting 50% of the population)), immunoassay (e.g., radio-linked immunoassay, enzyme- linked immunoassay), and single radio immunodiffusion assay.

[00122] Methods of direct challenge of vaccinated subject with infectious virus are known in the art. For comparison, groups of subjects (e.g., vertebrates) can be immunized by administrating one or more vaccines to be tested, at least one standard vaccine, and optionally at least one negative control, respectively. In one embodiment, different dosages of said vaccines to be tested and said standard vaccine can be tested in one comparison test. In one embodiment, said administrating can be intravenous, intraarterial, intramuscular, intracerebral, intracerebroventricular, intracardiac, subcutaneous, intraosseous infusion, intradermal, intrathecal, intraperitoneal, intravesical, intracavernosal, intranasal, transdermal, transmucosal, inhalational, intracisternal, epidural, or intravitreal. Groups of subjects (e.g, vertebrates, such as humans) are immunized at least once primarily. It is preferred that, a second round immunization is performed to boost immune responses in the subjects. Said groups of subjects are then challenged with infectious virus, and the effects are analyzed after a certain period of time. The results can be evaluated in various ways. In one embodiment, percentage of subjects showing infection symptoms can be compared between said vaccine to be tested and said standard vaccine. In anther embodiment, virus titer in the serum can be compared between subjects immunized with said vaccine to be tested and said standard vaccine. In another embodiment, ED₅₀ (vaccine dosage capable of protecting 50% of the population) can be compared between said vaccine to be tested and said standard vaccine.

[00123] Immunoassays are well known to one skilled in the art. Immunoassays are a diverse group of analytical techniques based on specific antibody/antigen interactions producing a measurable signal that can be related to the concentration of a compound in solution. In one embodiment, the antibody is monoclonal antibody. In another embodiment, the antibody is polyclonal antibody. In another embodiment, said immunoassays are competitive immunoassays. In another embodiment, said immunoassays are non-competitive immunoassays. In another embodiment, said immunoassays are heterogeneous assays wherein separation of bound and unbound components is required after the reaction has taken place (e.g., ELlSA). In another embodiment, said immunoassays are homogeneous assays wherein the binding reaction is measured in place without the separation of reaction components. Examples of commonly used immunoassays include, but are not limited to, radioimmunoassay (wherein one or more radioactive labels such as I^!2$, H³, C¹⁴ is utilized to emits radiation measured with a beta or gammacounter), enzyme immunoassay (wherein one or more enzymes are used to provide a measurable signal, e.g., glucose-6-phosphate dehydrogenase or β-galactosidase), Enzyme Linked Immunosorbent Spot (ELISI⁵OT) assay, fluorescence polarization immunoassay (FPIA), Kinetic Interaction of Microparticles in solution (KIMS), and lateral flow technology. Non-limiting examples of said enzyme immunoavssays are EMIT, Cloned Enzyme Donor Immunoassay (CEDl), and Enzyme-Linked Immunosorbent Assay (ELlSA). More detailed infoiτnation of immunoassays is described in David Wild, The immunoassay handbook, 2005, 3^rd Edition, Gulf Professional Publishing, ISBN 0080445268, 9780080445267. For comparison, serum of groups of subjects immunized with a vaccine to be test, at least one standard vaccine, and optionally at least one negative control respectively are analyzed in one or more immunoassay to decide the potency of said vaccine to be tested compared to one or more Standard vaccine. In another embodiment, responses of T-cells expressing antibody in immunized subjects can be compared to determine vaccine potency using Fluorescence-Activated Cell Sorter (FACS).

[00124] Single radial immunodiffusion (SRID) is used extensively for the quantitative estimation of antigens. The antigen-antibody precipitation is made more sensitive by the incorporation of antiserum in the agarose. Antigen (Ag) is then allowed to diffuse from wells cut in the gel in which the antiserum is uniformly distributed. Initially, as the antigen diffuses out of the well, its concentration is relatively high and soluble antigen-antibody adducts are formed. However, as Ag diffuses farther from the well the Ag-Ab complex reacts with more amount of antibody resulting in a lattice that precipitates to form a precipitin ring. Thus, by running a range of known antigen concentrations on the gel and by measuring the diameters of their precipitin rings, a calibration graph is plotted. Antigen concentrations of unknown samples, run on the same gel can be found by measuring the diameter of precipitin rings and extrapolating this value on the calibration graph. For comparison, serum of groups of subjects immunized with a vaccine to be test, at least one standard vaccine, and optionally at least one negative control respectively are analyzed in a SRlD assay to decide the potency of said vaccine to be tested compared to one or more standard vaccine.

Pharmaceutical or Vaccine Formulations and Administration

[00125] The pharmaceutical compositions useful herein contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to the vertebrate receiving the composition, and which may be administered without undue toxicity and a purified HA protein, an HA micelle comprising a purified HA protein, or a VLl' comprising a purified HA protein of the invention. As used herein, the term "pharmaceutically acceptable"' means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Phaπnacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans. These compositions can be useful as a vaccine and/or antigenic compositions for inducing a protective immune response in a vertebrate.

[00126] The invention also encompasses a pharmaceutically acceptable vaccine composition comprising a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein as described above. [00127] The invention also encompasses a kit for immunizing a vertebrate, such as a human subject comprising a purified HA protein, an HA micelle comprising a purified HA protein, OT a VLP comprising a purified HA protein as described above.

[00128] In one embodiment, the invention comprises an immunogenic formulation comprising at least one effective dose of a purified HA protein. In another embodiment, the invention comprises an immunogenic formulation comprising at least one effective dose of an HA micelle comprising a purified HA protein. Iu yet another embodiment, the invention comprises an immunogenic formulation comprising at least one effective dose of a VLP comprising a purified HA protein as described above.

[00129] The immunogenic formulation of the invention comprises a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein, and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (Mack Pub. Co. NJ. current edition). The formulation should suit the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans, preferably is sterile, non-particulate and/or non-pyrogenic.

[00130] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc,

[00131] The invention also provides for a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the vaccine formulations of the invention. In a preferred embodiment, the kit comprises two containers, one containing a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein, and the other containing an adjuvant. Associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00132] The invention also provides that the formulation be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of composition. In one embodiment, the composition is supplied as a liquid, in another embodiment, as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject.

[00133] In an alternative embodiment, the composition is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the composition. Preferably, the liquid form of the composition is supplied in a hermetically sealed container at least about 50 μg/ml, more preferably at least about 100 μg/ml, at least about 200 μg/mi, at least 500 μg/ml, or at least 1 mg/ml.

[00134] The invention also comprises a method of formulating a vaccine or antigenic composition that induces immunity to an infection or at least one disease symptom thereof to a mammal, comprising adding to the formulation an effective dose of a purified MA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein. In one embodiment, the infection is an influenza infection.

[0013S] While stimulation of immunity with a single dose is possible, additional dosages can be administered, by the same or different route, to achieve the desired effect. In neonates and infants, for example, multiple administrations may be required to elicit sufficient levels of immunity. Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against infections, e.g. influenza infection. Similarly, adults who are particularly susceptible to repeated or serious infections, such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may require multiple immunizations to establish and/or maintain protective immune responses. Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.

[00136] Methods of administering a composition comprising a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein (e.g. vaccine and/or antigenic formulations) include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intravenous and subcutaneous), epidural, and mucosal {e.g., intranasal and oral or pulmonary routes or by suppositories). In a specific embodiment, compositions of the present invention are administered intramuscularly, intravenously, subcutaneously, transdermally or inrradermally. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.) and may be administered together with other biologically active agents. In some embodiments, intranasal or other mucosal routes of administration of a composition of the invention may induce an antibody or other immune response that is substantially higher than other routes of administration. In another embodiment, intranasal or other mucosal routes of administration of a composition of the invention may induce an antibody or other immune response that will induce cross protection against other strains of influenza. Administration can be systemic or local.

[00137] In yet another embodiment, the vaccine and/or immunogenic formulation is administered in such a manner as to target mucosal tissues in order to elicit an immune response at the site of immunization. For example, mucosal tissues such as gut associated lymphoid tissue (GALT) can be targeted for immunization by using oral administration of compositions which contain adjuvants with particular mucosal targeting properties. Additional mucosal tissues can also be targeted, such as nasopharyngeal lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT).

[00138] Vaccines and/or immunogenic formulations of the invention may also be administered on a dosage schedule, for example, an initial administration of the vaccine composition with subsequent booster administrations. In particular embodiments, a second dose of the composition is administered anywhere from two weeks to one year, preferably from about 1, about 2, about 3, about 4, about 5 to about 6 months, after the initial administration. Additionally, a third dose may be administered after the second dose and from about, three months to about two years, or even longer, preferably about 4, about 5, or about 6 months, or about 7 months to about one year after the initial administration. The third dose may be optionally administered when no or low levels of specific immunoglobulins are detected in the serum and/or urine or mucosal secretions of the subject after the second dose. In a preferred embodiment, a second dose is administered about one month after the first administration and a third dose is administered about six months after the first administration. In another embodiment, the second dose is administered about six months after the first administration. In another embodiment, the compositions of the invention can be administered as part of a combination therapy. For example, compositions of the invention can be formulated with other immunogenic compositions, antiviral s and/or antibiotics. [00139] The dosage of the pharmaceutical composition can be determined readily by the skilled artisan, for example, by first identifying doses effective to elicit a prophylactic or therapeutic immune response, e.g., by measuring the serum titer of virus specific immunoglobulins or by measuring the inhibitory ratio of antibodies in serum samples, or urine samples, or mucosal secretions. The dosages can be determined from animal studies. A non-limiting list of animals used to study the efficacy of vaccines include the guinea pig, hamster, ferrets, chinchilla, mouse and cotton rat. Most animals are not natural hosts to infectious agents but can still serve in studies of various aspects of the disease. For example, any of the above animals can be dosed with a vaccine candidate, e.g. VLPs of the invention, to partially characterize the immune response induced, and/or to determine if any neutralizing antibodies have been produced. For example, many studies have been conducted in the mouse model because mice are small size and their low cost allows researchers to conduct studies on a larger scale.

[00140] In addition, human clinical studies can be performed to determine the preferred effective dose for humans by a skillet! artisan. Such clinical studies are routine and well known in the art. The precise dose to be employed will also depend on the route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal test systems.

[00141] As also well known in the art, the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and as such, adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation. The inclusion of any adjuvant described in Vogel ei al., "A Compendium of Vaccine Adjuvants and Excipients (2^nd Edition)," herein incorporated by reference in its entirety for all purposes, is envisioned within the scope of this invention.

[00142] Exemplary, adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CCJP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/IVeen 80 emulsion also is contemplated. MF-59, Novasomes^®, MHC antigens may also be used.

[00143] In one embodiment, the purified ILA protein or HA micelle comprising a purified HA can be used as an adjuvant to boost a vaccine comprising a VXP. In one embodiment, the VLP comprises an HA protein.

[00144] In one embodiment of the invention the adjuvant is a paucilamellar lipid vesicle having about two to ten bilayers arranged in the form of substantially spherical shells separated by aqueous layers surrounding a large amorphous central cavity free of lipid bilayers. Pauciiamellar lipid vesicles may act to stimulate the immune response several ways, as non-specific stimulators, as carriers for the antigen, as carriers of additional adjuvants, and combinations thereof. Paucilamellar lipid vesicles act as non-specific immune stimulators when, for example, a vaccine is prepared by intermixing the antigen with the preformed vesicles such that the antigen remains extracellular to the vesicles. By encapsulating an antigen within the central cavity of the vesicle, the vesicle acts both as an immune stimulator and a carrier for the antigen. In another embodiment, the vesicles are primarily made of nonphospholipid vesicles. In other embodiment, the vesicles are Novasomes^®. Novasomes^® are paucilamellar nonphospholipid vesicles ranging from about 100 ran to about 500 nm. They comprise Brij 72, cholesterol, oleic acid and squalene. Novasomes have been shown to be an effective adjuvant for influenza antigens (see, U.S. Patents 5,629,021 , 6,387,373, and 4,91 1,928, herein incorporated by reference in their entireties for all purposes).

[00145] The compositions of the invention can also be formulated with ''immune stimulators." These are the body's own chemical messengers (cytokines) to increase the immune system's response. Immune stimulators include, but not limited to, various cytokines, Iymphokines and chemokines with immunostimulatory_> immunopotenliating, and pro-inflammatory activities, such as interleukins (e.g., IL-I , IL-2, IL-3, 1L-4, IL- 12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other imrnunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1 ; B7.2, etc. The immunostimulatory molecules can be administered in the same formulation as the compositions of the invention, or can be administered separately. Either the protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect. Thus in one embodiment, the invention comprises antigenic and vaccine formulations comprising an adjuvant and/or an immune stimulator. Methods of Stimulating an Immune Response

[00146] The purified HA proteins of the invention are useful for preparing compositions that stimulate an immune response that confers immunity or substantial immunity to infectious agents. Both mucosal and cellular immunity may contribute to immunity to infectious agents and disease. Antibodies secreted locally in the upper respiratory tract are a major (actor in resistance to natural infection. Secretory immunoglobulin A (slgA) is involved in the protection of the upper respiratory tract and serum IgG in protection of the lower respiratory tract. The immune response induced by an infection protects against reinfection with the same virus or an antigenically similar viral strain. For example, influenza undergoes frequent and unpredictable changes; therefore, after natural infection, the effective period of protection provided by the host's immunity may only be effective for a few years against the new strains of virus circulating in the community.

[00147] Thus, the invention encompasses a method of inducing immunity to infections or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

[00148] In one embodiment, the invention comprises a method to induce immunity to influenza infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein. In another embodiment, the invention comprises a method to induce immunity to influenza infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of an HA micelle comprising a purified HA protein. In yet another embodiment, the invention comprises a method to induce immunity to influenza infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of influenza VLP, wherein the VLPs comprise a purified HA protein.

[00149] The invention also encompasses inducing immunity to an infection, or at least one symptom thereof, in a subject caused by an infectious agent, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

[00150] Compositions of the invention can induce substantial immunity in a vertebrate (e.g. a human) when administered to the vertebrate. The substantial immunity results from an immune response against compositions of the invention that protects or ameliorates infection or at least reduces a symplom of infection in the vertebrate. In some instances, if the vertebrate is infected, the infection will be asymptomatic. The response may not be a folly protective response. In this case, if the vertebrate is infected with an infectious agent, the vertebrate will experience reduced symptoms or a shorter duration of symptoms compared to a non-immunized vertebrate.

[00151] In one embodiment, the invention comprises a method of inducing substantial immunity to influenza virus infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein. In another embodiment, the invention comprises a method of vaccinating a mammal against influenza comprising administering to the mammal a protection-inducing amount of purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

[00152] The invention also encompasses a method of inducing substantial immunity to an infection, or at least one disease symptom in a subject caused by an infectious agent, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein.

[00153] In another embodiment, the invention comprises a method of inducing a protective antibody response to an infection or at least one symptom thereof in a subject, comprising administering at least one effective dose of a purified HA protein, an HA micelle comprising a purified HA protein, or a VLP comprising a purified HA protein as described above.

[00154] As used herein, an "antibody" is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG. IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light-' (about 25 kD) and one "heavy" chain (about 50-70 kD). The N -terminus of each chain defines a variable region of about 100 to UO or more amino acids primarily responsible for antigen recognition. Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. [00155] In one embodiment, the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose of a purified HA protein. In another embodiment, the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose an HA micelle comprising a purified HA protein. In yet another embodiment, the invention comprises a method of inducing a protective cellular response to influenza infection or at least one disease symptom in a subject, comprising administering at least one effective dose a VLP, wherein the VLP comprises a purified HA protein as described above.

[00156] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figure and the Sequence Listing, are incorporated herein by reference for all purposes.

EXAMPLE

Example 1

Expressing Recombinant Influenza A HA Protein of A/Cal/04/09 HlSNl Virus

[003] Sequence of influenza A ϊϊA protein of A/Cal/04/09 HlNl virus showed below was expressed in a baculovirus expression system.

Hemagglutinin, HA, from Influenza A virus (A/CaJifoπiaia/04/2009 HlNl)

[004] Once tlie desired construct was confirmed and purified (pFastBac™), one vial of MAX Efficiency® DHlOBac™ competent cells was thawed on ice. Approximately 1 ng (5 μl) of the desired pFastBac™ construct plasmid DNA was added to the cells and mixed gently. The cells were incubated on ice for 30 minutes. This was followed by heat-shock of the cells for 45 seconds at 42° C without shaking. Next, the tubes where immediately transferred to ice and chilled lor 2 minutes. Subsequently 900 μl of room temperature S.O.C. Medium was added to each tube. The tubes were put on a shaker at 37°C at 225 rpm for 4 hours. For pFastBac™ transformation, 10-fold serial dilutions of the cells (10-1. 10-2 and 10-3) was prepared using S.O.C. medium. Next, 100 μl of each dilution was plated on an LB agar plate containing 50 μg/ml kanamycin. 7 μg/ml gentamicin, 10 μg/ml tetracycline. KM) μg^/nul Bluo-gal, and 40 μg/ml IPTG . The plates were incubated for 48 hours at 37° C. White colonies where picked for analysts. Bacmid DMA from above was made for construct expressing HA of Influenza A/Cal/04/09 and was isolated to make baculovirus #736 (primary virus infected with transfection medium).

[00157] Next, 10 liters WCBlOl Si9 insect cells were infected with baculovirus expressing HA of Influenza A/Cal/04/09 with plaque eluate and incubated 68-72 hours. Sf9 cells of MOl~0.2 were harvested by centrifuge at 4000rpm, and the cell pellet was washed with IxPBS for three times. Pelleted cells were equally divided to 5 tubes, each contains about 25 gram cells pelleted from about 2 liters ceil culture. Four tubes were stored at -70°C for future use, and 1 tube of cells was stored at -20°C for purification on the following day.

Example 2

Purifying Recombinant Influenza HA Protein

[00158] Ic this example, purification method of influenza HA protein was demonstrated by purifying HA protein derived from influenza B/Brisbane/60. Tubes at -20°C containing Sf9 cells transfected with baculovirus expressing influenza HA was thawed at room temperature. Since HA proteins are expressed intracellυlarly, cell lysis and extraction for recovery are required. Bacυiovirus protein gp64 is the most common contaminant that was observed to cυ-purirled with HA protein. Varied results were observed among different HA when apply the same chromatography procedure. The purification method can be evaluated and, if necessary, modified for each individual HA due to differences in physical/chemical characteristics.

[00159] Materials and equipment for purification arc listed below:

Chemicals (All chemicals must be at least IIS P grade υr equivalent, preferably multi- compendial when available):

• Tn s (base): Tromethamine, USP, multi -compendial, J.T. Baker, Cat# 4102-05,

FW121.14 • NaCl: Sodium chloride USP, multi-compendial, J.T. Baker, Cat# 3627-07, FW 58.44

• NaOH: Sodium hydroxide Pellets, N.F. multi-compendial, J. T. Baker, Cat# 3718-05, FW 40.00

• CaC12: CaJcium chloride dihydrate, granular, USP, FCC, Mallinckrodt, Cat# 4616-04, FW 147.01

• MnC12: Manganese chloride tetrahydrate, ACS grade, Sigma-Aldrich, Cat# 221279, FW 197.91

• NP-9: Tergitol NP-9, Aidrich, Cat# 521078

• l,eupeptin: Leupeptin Hemisulfate Salt, Sigma, Cat# L2884

• Methyl-α-D-Mannoρyτanoside: Fisher, Cat// BP2530

• Sodium Phosphate Monobasic: NaH2PO4 raonohydrate, USP, J. T. Baker, Cat# 3802- 01 , FW ( 37.99

• 1 ON NaOH : J.T. Baker, Cat# 5000-03

• 6N HCl: J. T. Baker, Cat# 0327-02

• DPBS: w/o Ca, Mg, GlBCO, Cat# 14190

HPLC, column, resins and filtration equipment;

• AKTA Explorers equipped with Unicorn Software V5.1. or other equivalent HPLC

• Empty columns XK 26/40 (or XK5O/3O), XK26x70, two of XKI 6/20, from GE Healthcare

• Fractogel® EMD TMAE Hicap (M) resin, EMD Chemicals, Inc. Cat# 1.10316.0500

• Lentil lectin Sepharose 4B, G E, cat# 17-0444-01

• Sephadex G-25 Medium: GE HealthCare, Cat# 17-0033-02

• Macro-Prep Ceramic Hyroxyapatite Type 1 (40μm) revsin, BIO-IlAD, Cat# 157-0040, or pre-packed column "Bio-Scale Mini CHI-™ Type I, 40μm Cartridge (Ix5mi)'\ BIO-RAD, cat# 732-4322.

• Amicon 8050 Series Stir Cells, Millipore, Cat# 5122

• Ultrafiltration membrane, Regenerated cellulose, Dia. 44.5mm, NMWL: 30K.

Millipore, Cat# PLTK04310

• Slide- A-Lyzer Dialysis Cassette, 2OK MWCO, 12-30 ml, Thermo Scientific (PIERCE), Prod # 66030 [00160] To make buffers and solutions for purification, WFi or KOfDl water as supplied by the in-house system was used for all preparation. Volumes were measured using glass graduated cylinders. AU weighing was ± 1% of stated values. ^'Hie pH of the solution was adjusted to a target ±0.1 of the required pH. Final pH specification was ±0.2 allowing for variation between meters, temperature and other factors. Each buffer/solution was filtered through 0.22 μm cellulose acetate (or polyvinylidene, polyethersulfone) filters. Freshly made buffers/solutions were prepared for each run to lower endotoxin level in the product. AU buffers/solutions were prepared and stored at room temperature (~25°C). Buffers and solutions used in purification are shown below:

IM THs, pH 8.0 Stock Solution (II,)

Dissolve 121 g of Trometharnine in approximately 800 mi of water. Mix solution using a magnetic stirrer. Adjust the pH to 8.0 ± 0.1 , Solution must be allowed to come to room temperature {20 to 25^δC) before final adjustment, QS to 1000 ml.

5 M NaCl Stock Solution (2L)

Add 584.4 g Sodium Chloride to approximately 1500 ml of water. Mix and dissolve with a magnetic stirrer. QS to 2000 ml in a glass graduated cylinder.

1 M NaOH (IL)

Add 40.0 g of Sodium Hydroxide to approximately 800 ml water. Mix and dissolve using a magnetic stirrer. QS to 1000 ml using a graduated cylinder. Filter using a 0.2 micron PES filter.

1 M Calcium Chloride (200ml)

Dissolve 29.4 g calcium chloride dihydrate to 180 ml with water. QS to 200ml.

I M MnCl₂ (IOOmI)

Dissolve 19.79 g manganese chloride tetrahydrate to 80ml water, QS to 100 ml.

10% NP-9 (VAO (100ml)

Add 10.6 g of Tergitol NP-9 (Tergilol NP-9 density 1.06 g/mL) to water, QS 100 ml.

Leupeptin stock (1 mg/mL)

Suspend 25 mg leupeptin in 25 ml of TMAE equilibration buffer.

Aliquot the solution into 1 ml cryovial and stored at -2O°C.

0.5M Sodium Phosphate Buffer, pH 6.8 (IL)

Dissolve 69.0 g NfIH₂POj monohydrate in 800ml water. Adjust pH with ION NaOH to 6.8. QS to 1000 ml. Extraction Buffer (30 mM Tris, pH 8.0, 100 mM NaCl, 1% NP-9, 1 niM

Ca⁺^) (IL)

30 ml I M Tris, pH 8.0

20 ml 5 M NaCl

10.6 g of Tergitol NP-9 (Tergitol NP-9 density 1.06 g/mL)

1 ml 1 M Calcium Chloride

QS to 1000 ml

TMAE Equilibration Buffer (30 mM Tris, pH 8.0, 100 mM NaCI, 0.02% NP-9) (3L)

90 ml 1 M Tris, pH 8.0

60 ml 5 M NaCl

6 ml 10% NP-9

QS to 3000 ml

TMAE EIution buffer (30 niM Tris, pH 8.0, IM NaCl, 0.02% NP-9) (IL)

3O mI l M Tris, pH 8.0

200 ml 5 M NaCl

2 ml of 10% NP-9

QS to 1000ml

Lentil lectin equilibration buffer (30 mM Tris, pH 8, 500 mM NaCl, 0.02 % NP- 9, 1 mM CaCIi, 1 niM MnCh) (1000ml)

3O mI lM Tris, pH 8.0

100 ml 5M NaCl

2 ml 10% NP-9

1 ml IM CaCl₂

I mI lM MnCl₂

QS to 1000ml

Lentil lectin elution buffer (30 mM Tris, pH 8_S 500 mM NaCl, 0.02 % NP-9, 1 mM CaCl₂, 50OmM Methyl-α-D-mannopyranoside) (250ml)

7.5 mi l M Tris, pH 8.0

25 ml 5M NaCl

0.5 mi 10% NP-9

0.25 ml IM CaCl₂

24.3 g Methyl -α-D-mannopyranoside

QS to 250ml

CHT™ equilibration buffer (10 mM sodium Phosphate, pH 6.8, 50 mM NaCl, 0.02% NP-9) 3L

60 ml 0.5M NaPi pH 6.8

30 ml 5 M NaCl

6 mi l 0% NP-9

Add water to -2.8L

Adjust pH to 6.8

QS to 3000 ml

CHT™ Elution (250 mM sodium Phosphate, pH 6.8, 50 mM NaCI, 0.02% NP-9) 500 ml 250 ml 0.5M NaPi pH 6.8

5 ml 5M NaCl

l ml lO% NP-9

Add water to ~450 ml

Adjust pH to 6,8

QS to 500 ml

[00161] Columns used in the purification were packed as described below:

L Pack TMAE column per EMD instructions (EMD Fractogel® Instruction booklet). Pack at 10ml/min if using a XK 26/40 column. Pack at 65ml/inin if use a XKSO/30 column (so not to exceed the pressure limit of the column).

2. Pack Lentil lectin column per GE instructions (GE Health Lentil Lectin Instruction booklet). Use an XK 16/20 column and pack at a flow rate of 70«^'% to a height of 5- 10 cm ( 10-20 ml).

3. Prepare G-25 (2.6x50) per GE instructions using XK 26/70 column. The column volume must be 3-5 times of the loading volume.

4. Pack a 10 ml (1.6x5cm) Ceramic Hydroxyapitite column per BioRad Instructions (BioRad CHT™ Instruction booklet). Alternatively, a 5ml prepacked column (from BioRad) can be used.

1_.00.162] Pelleted Sfl) cells containing recombinant influenza HA were suspended in approximately 800-1000 ml of extraction buffer (see above). The suspension was then allowed to stir for 1 hour at room temperature using a magnetic mixer, and centrifuged at 8000 rpm in GS-4 rotor for 30 nuns. The supernatant containing recombinant influenza HA was then filtered using 0.45 micron cellulose acetate or other equivalent filters. The supernatant was first run though Fractogel® EMD TMAE (M) column.

[00163] Fractogel EMD TMAE (M) column (150-40OmI) column was sanitized before use as described below:

1. One column volume (CV) IN NAOH was used to clean the column at flow rate of 30-50 cn/hr followed by 1 column volume of water.

2. The column was then pre-eqυilibrated with 1 CV of elution buffer prior to the equilibration.

3. The column was then equilibrated with enough amount of TMAE EQ buffer at flow rate of lOOcm/hr until pH and conductivity reached the same as the EQ buffer.

4. One CV filtered extraction buffer was applied to equilibrate the just prior to the run. [00164] Filtered supernatant was then loaded on the column at flow rate of lOOcm/hr. The column was washed with 2-3 column volume equilibration butler. Flow through and washing solution were collected and as single bulk fractions. A 10 column volume gradient of 0% to 100% elution buffer was used to elute the column. 50 ml fractions were collected. The flow through and gradient elution fractions were examined by SDS-PAGE (Coomassie blue staining ami Western blot). Figure 1 showed the TMAE chromatogram (Figure 1 a) and SDS PAGE gel scan (Figure Ib) for influenza HA of B/Brisbane 60. Eluted fractions containing HA were pooled together for further purification.

[00165] The HA pool from TMAE column was further purified by lentil lectin (LL) affinity chromatography. A 10-20 ml Ll., column was equilibrated with sufficient (>3 CV) LL EQ buffer at flow rate of 50 cm/hr through the entire run. The hA pool to be loaded was adjusted to 1 mM calcium and manganese by adding 1 ml of 1 M MnCl₂ and 1 mL of 1 M CaCl? per liter. The HA pool from TMAB column was then loaded onto the LL column. The column was washed with 2-3 CV of EQ buffer. HA was the eluted from the column with lentil lectin elution buffer collecting 5ml fractions. Elution fractions were examined on SDS-PAGE (Coomassie and western blot). Fractions containing HA were then pooled together. Figure 2 showed the lentil lectin chromatography (Figure 2a) and SDS PAGE gel scan (Figure 2b) for influenza HA of B/Brisbane 60. There were two bands observed on the gel, one was HA, another one is gp64 from baculovirus.

[00166] The HA pool from lentil lectin affinity chromatography was desalted with a G25 Desalting Chromatography or Dialysis before further purified with CHT™ column. The pool of HA fractions from the LL column must be buffer exchanged to the EQ buffer of CHT™ column prior to loading on to CHT™ column. Either Sephadex G25 chromatography or dialysis can be used. CHT EQ buffer was used for G25 desalting step. G25 column was equilibrated with CHT™ EQ buffer first. HA pool was then loaded to G25 column at flow rate of 100 cm/hr. 50 ml fractions were collected and pooled at the protein peak (A280). Alternatively, 20-30K MVVCO membrane dialysis bag/tubing (Slide-A-Lyzer Dialysis cassette) or Amicon Stir Cells were used. Two buffer exchange volumes for dialysis (each against buffer at >50 fold sample volume) were recommended for more than 6 hours (each) at 4°C.

[00167] Desalted protein peak containing influenza HA was further purified with CHT™ column. A CHT column (Type 1, 5-10 ml) was equilibrated with CHT^rM EQ buffer. Flow rate through EQ, load and wash was at 0.5-1.0 CV/min (2.5-5.0 ml/min for a 5 ml column). The protein peak from the G-25 column or solution after dialysis was loaded onto the column. The cohimn was washed with 2-3 CV of EQ buffer. Protein was eluted with 10-15 CV gradient from 0 to 100% of elution buffer at flow rate of 0.5CV/min with fraction size at <!/2 CV. The flow through and elution fractions were analyzed on SDS-PAGE. Figure 3 showed B/Bris HA CHT™ chromatogram (Figure 3a) and SDS-PAGE (Figure 3b).

[00168] Pandemic H5N1 HA protein was observed in the FT of CHT™ chromatography, while others, including H3N2 HA, HlNl HA and B strain HA, were observed in late fractions of the gradient elution. Impurity (gp64 ) is eluled at beginning of the phosphate gradient (—100 mM phosphate). Fraction pooling must be performed carefully, and yield is typically sacrificed for good purity.

[00169] HA protein can be concentrated and dialyzed extensively using a Millipore Stirred Cells with 20-30K MWCO membrane against DPBS buffer. A minimum of three additions of 10-fold buffer volume is needed for dialysis. Final HΛ protein concenration at >0.5 mg/ml is preferred. Sterile Filtration is performed using a 0.2 micron SFCA (or PVDF) filter. HA yield is approximately 5-10 mg (from 2L cell culture).

Example 3

Purifying Recombinant Influenza OA Protein Derived from Influenza

A/California/04/09

[00170] In this example, purification method of influenza HA protein was demonstrated by purifying HA protein derived from influenza A/Cal/04/09. Tubes at -20°C containing Sf9 cells transfected with baculovirus expressing influenza HA derived from influenza A/Cal/04/09 was thawed at room temperature. It was estimated that the wet cells weight was 24.9 gram. Cell pellet was re-suspended with 400 ml 25 mM TrisCl pH 8.0, 50 mM NaCl, 1% NP9, 2 ug-'ml Leυpeptin, 16 mi lysis buffer per gram cell, and stirred at room temp for 70 min. The solution was then centrifuged at 10,000 g, SLA 1500 rotor for 30 min at 15 C. 400 ml supernatant was taken for purification.

[00171] Freshly packed 383 ml Fractogel TMAE (M) column with 5 cm diameter. 19.5 cm height, was cleaned with 0.5 N NaOH. Flow rate was 30 ml/min for packing and 20 ml/min for running. The column was equilibrated with 25 mM TrisCl pH 8.0, 50 mM NaCl, 0.02% NP9. 400 ml supernatant solublized with 1% NP9 was loaded through. The column was then washed with 3 CV with buffer A (25 mM 1 Vis pH 8.0, 50 mM NaCl, 0.02% NP9).

(00J 72] 500 ml flow through was collected. Protein was eluted with 2 CV with 25 % buffer β (25 mM Tris pH 8.0, 1000 mM NaCl, 0.02% NP9), 300 mM NaCl, and 200 ml elute was collected. Protein was further eluted with 2 CV 50% buffer B, 525 mM NaCl, and 200 ml elυte. Protein was finally eluted with 2 CV with 100% buffer B. 1000 mM NaCl, and 150 ml elute was collected. Analysis showed that all HA flew though the TMAK column, and almost all GP64 bound to the column (data not shown).

[00173] Column was cleaned subsequently with 2 CV 0.5 N NaOH, 3 CV 25 mM Tris pH 8.0, 1000 mM NaCl, 0.02% NP9 , and 25 mM Tris pH 8.0, 50 mM NaCK 0.02% NP9.

[00174] Freshly packed 25 ml lentil lectin sepharose 4B affinity column with 2.6 cm diameter, 4.7 cm height was kept in 20% EtOH at 4 C. Flow rate was 6.25 ml/min for packing and 5 ml/min for running. The column was equilibrated with 25 mM TrisCJ pH 8.0, 500 mM NaCl₅ 1 mM MnCI2, 1 mM CaC12, 0.02% NP9 to recharge column with metal, then equilibrated with 25 mM TrisCl pH 8.0, 500 mM NaCl, 0.02% NP9. 500 ml TMAE flow through in 1% NP*⁾ was loaded through. The column was then washed with 10 CV with buffer C (25 mM TrisCl, pH 8.0, 500 mM NaCl, 0.02% NP9), and protein was eluted with 10 CV with 100% buffer D (25 mM TrisCl pH 8.0, 500 mM NaCl, 0.02% NP9, 500 mM methyl- alpha-D-mannopyranoside). 60 ml elution was collected. Column was cleaned in 20% EtOH and at 4 °C. Analysis showed that HA bound and was eluted from lentil lectin column, and the purity reached 97% (data not shown).

[00175] Previously packed 470 ml Sephadex G25 column with 5 cm diameter was cleaned with 0.5 N NaOH. NaOH was removed with 25 mM TrisCl pH 8.0 500 mM NaCl. Flow rate was 15 mlmin for running. The column was equilibrated with GibcoΦ PBS, Lot 548528. 60 πύ Lectin column elute was loaded. Without washing, the protein was eluted with 1.5 CV buffer E (IX PBS, pH 7.2). Analysis showed that HA was eluted in the void peak. Fractions 1 and 2 were combined (total 75 nil) and cleaned with 0.2 μm sterile filtration (corning filter lot #00709002). Sample was used for quality control analysis by SDS-PAGE gel (Coomassie blue stain and Western blot with influenza A/New CaI Hl antibody). As it was shown in Figure 4, HA protein with high purity (>97%) was isolated, and the yield was about 1 1 mg liter of cell culture. Table 1 below showed detailed quality control analysis results.

Table 1. Quality Control Analysis Results

Example 4

Expressing Chimeric influenza A HA and NA using Influenza matrix protein to assemble VLPs

[00176] The sequences below are recombinant HA, recombinant NA, and recombinant Ml derived from influenza A/Californai/04/09, which were expressed to assemble influenza A/Californai/04/09 VUPs. These sequences are co-expressed in a baculovirus expression system to produce chimeric VLPs that express influenza HA antigens on the surface of VLPs.

Hemagglutinin, HA, from Influenza A virus (A/Caiifomata/04/2009 Hl Nl )

[00177] Next, VLPs were harvested and isolated from the supernatant by centrifugation and by a discontinuous sucrose step gradient. The fraction comprising the VLPs was collected from the top of the gradient. The VLPs isolated from the sucrose gradient were analyzed by SDS-PAGE and western blot. VLPs were turther purified as described below.

[00178] The concentrated, diafiltered VLPs were furthered purified on a 20% to 60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaCl by centrifυgation at 6,500 x g for 18 hours at about 4° C to about 10° C. Usually VLPs will form a distinctive visible band between about 30% to about 40% sucrose or at the interface (in a 20% and 60% step gradient) that can be collected from the gradient and stored. This product was diluted to comprise 200 mM of NaCl in preparation for the next step in the purification process. This product contained VLPs and might contain intact baculovirus particles.

[00179] Further purification of VLPs was achieved by anion exchange chromatography, or 44% isopycnic sucrose cushion ceritrifugation. In anion exchange chromatography, the sample from the sucrose gradient (see above) was loaded into column containing a medium with an anion (e.g. Matrix Fractogel EMD TMAE) and eluded via a salt gradient (from about 0.2 M to about 1.0 M of NaCl) that can separate the VLP from other contaminates (e.g. baculovirus and DNA/RNA). hi the sucrose cushion method, the sample comprising the VLPs was added to a 44% sucrose cushion and centrifuged for about 18 hours at 30,000 g. VLPs formed a band at the top of 44% sucrose, while baculovirus precipitated at the bottom and other contaminating proleins stay in the 0% sucrose layer at the top. The VLP peak or band was collected.

[00180] Purified HA protein and purified VLPs were submitted to FDA for vaccine potency assay.

Example 5

Vaccine Potency Assay

[00181] Purified recombinant HA of influenza A/California/04/09 (49 μg/ml) and purified

VLPs (Lot 747.3, 0.35 mg/ml) were used in a vaccine potency assay.

[00182] To test the potency of VLP vaccine, sheep were immunized with mixture of VLPs and recombinant HA of influenza A/California/04/09. Priming immunization was done using

0,2 ml VLP containing 9.8 ug HA together with 50 ug recombinant HA (rHA), and boost immunization was done using 0.1 m! VLP containing 4.9 ug IiA together with 30 ug rHA.

Sheep antiserum after co-immunization with A/California/04/09 H lNl VLP and recombinant

HA was harvested and used to make agarose gel for SRID assay. A non-limiting exemplary protocol of SRID assay is demonstrated below: 1. Prepare 10 ml of 1.0% agarose (0.1 g/ 1 OmI) in 1 X assay buffer (Phosphate buffered saline) by heating slowly till agarose dissolves completely. Take care not to scorch or troth the solution.

2. Allow the molten agarose to cool to 55°C.

3. Add 120 ml of antiserum to 6 ml of agarose solution. Mix by gentle swirling for uniform distribution of antibody.

4. Pour agarose solution containing the antiserum onto a grease free glass plate set on a horizontal surface. Leave it undisturbed to form a gel.

5. Cut wells using a gel puncher, using the template provided.

6. Add 20 ml of the given standard antigens and test antigens to the wells.

7. Keep the gel plate in a moist chamber (box containing wet cotton) and incubate overnight at room temperature.

8. Mark the edges of the circle and measure the diameter of the ring. Note down your observations.

9. Plot a graph of diameter of ring (on Y-axis) versus concentration of antigen (on X- axis) on a semi-log graph sheet.

10. Determine concentration of unknown by reading the concentration against the ring diameter from the graph.

[00183] Series diluted Egg-derived HA 2009 HlNl and egg-derived HA from former swine flu, together with commercial rHA, rHA (NVAX), and VLP (NVAX) were inoculated into wells on the agarose gel containing antiserum of sheep immunized with mixture of VLPs and recombinant HA of present invention which were derived from A/Califomia/04/09 HlNl, The SRlD results are shown in Figure 5. High levels of anlibody against HA of HlNl was stimulated in the sheep serum (column 1) (egg-derived HA 2009 Hl Nl).

[00184] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[00185] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. [00186] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

CLAIMS:

1. A purified hemagglutinin (HA) protein, wherein said HA protein is purified by a method comprising the steps of:

(i) Expressing said HA in a host cell;

(ii) Extracting said HA from said host cell, wherein said HA is soluble in an extraction buffer;

(iii) Purifying said HA from an extraction buffer using ion exchange chromatography, wherein a pool of tractions containing said HA is produced;

(iv) Purifying said HA from said pool in step (iii) using affinity chromatography, wherein a second pool of fractions containing said HA is produced;

(v) Desalting said second pool in step (iv);

(vi) Purifying said HA from the desalted pool of step (v) using hydroxyapatite chromatography, wherein a third pool of fractions containing said HA is produced; and optionally

(vii) Concentrating, dialyzing, and sterilizing said third pool of fractions containing HA.

2. The purified HA of claim 1, wherein die ion exchange chromatography in step (iii) is an anion exchange chromatography.

3. The purified HA of claim 2, wherein the anion exchange chromatography is a TMAE anion exchange chromatography.

4. The purified HA of any of claims 1 -3, wherein the affinity chromatography in step (iv) is a lentil lectin affinity chromatography.

5. The purified HA of any of claims 1-4, wherein the desalting step (v) is conducted with desalting chromatography.

6. The purified HA of claim 5, wherein desalting chromatography is performed using a desalting column packed with Sephadex™ G-25.

7. The purified HA of any of claims 1-6, wherein the desalting step (v) is conducted with dialysis.

8. The purified HA of claim 7, wherein the dialysis is performed using a dialysis bag, tubing, or a stirred cell comprising a selectively permeable membrane.

9. The purified HA of claim 8, wherein the selectively permeable membrane has a MWCO of about 20 to about 30 JcD.

10. The purified HA of any of claims 1-9, wherein the hydroxyapatite chromatography in step (vi) is performed using a column packed with synthetic hydroxyapatite.

1 1. The purified HA of claim 10, wherein the synthetic hydroxyapatite is ceramic hydroxyapatite.

12. The purified HA of any of claims 1-11, wherein the HA is concentrated and dialyzed in a stirred cell with selectively permeable membrane in step (vii).

13. The purified HA of any of claims 1-12, wherein the HA protein is selected from the group consisting of Hl, H2, H3, H4, H5, H6, H7, H8, H9, HlO, Hl 1 , H12, H13, H14, 1115, and Hl 6.

14. The purified HA of any of claims 1-12, wherein the HA protein is derived from an influenza virus strain selected from the group consisting of avian influenza virus strain and a mammalian influenza strain.

15. The purified HA of claim 14, wherein the HA is derived from an influenza Hl Nl virus strain.

16. The purified HA of claim 15, wherein the HlNl virus strain is selected from the group of A/Califomia/04/09, A/New Caledonia/20/ 1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.

17. The purified HA protein according to any of claims 1-16, wherein said host cell is a eukaryotic cell.

18. The purified HA protein according to claim 17, wherein said eukaryotic cell is an insect cell.

19. The purified HA protein according to claim 18, wherein said insect cell is an SrP cell.

20. A purified micelle comprising one or more purified HA proteins of any of claims 1- 19.

21. A virus-like particle (VLP) comprising a purified HA protein of any of claims 1-19.

22. The VLP of claim 21, further comprising an influenza matrix (Ml) protein.

23. The VLP of claim 22, wherein said Ml protein is derived from an avian strain of influenza virus.

24. The VLP of claim 22, wherein said M 1 protein is derived from a mammalian strain of influenza virus.

25. The VLP of any of claims 21 -24, further comprising an influenza neuraminidase (NA) protein.

26. The VLP of any of claims 21-25, further comprising an influenza matrix (M2) protein.

27. The VLP of any of claims 21-26, further comprising an influenza nυcleocapsid (NP) protein.

28. The VLP of any of claims 21-27, wherein the VLP is expressed in a eukaryotic cell under conditions which permit the formation of VLPs.

29. The VLP of claim 28, wherein the eυkaryotic cell is selected from the group consisting of yeast, insect, amphibian, avian, mammalian, or plant cells.

30. An immunogenic composition comprising a purified HA protein of any of claims I- 19.

31. An immunogenic composition comprising a purified micelle according to claim 20.

32. An immunogenic composition comprising a VLP according to any of claims 21-29.

33. A pharmaceutically acceptable vaccine composition comprising a purified HA protein of any of claims 1-19, wherein the purified HA protein is capable of eliciting an immune response in a host.

34. A pharmaceutically acceptable vaccine composition comprising a purified micelle according to claim 20, wherein the micelle is capable of eliciting an immune response in a host.

35. A pharmaceutically acceptable vaccine composition comprising a VLP according to any of claims 21-29, wherein the VLP is capable of eliciting an immune response in a host.

36. A kit for immunizing a human subject against a viral infection comprising a purified HA protein of any of claims 1 -19.

37. A kit for immunizing a human subject against a viral infection comprising a purified micelle according to claim 20.

38. A kit for immunizing a human subject against a viral infection comprising a VLP according to any of claims 21-29.

39. The kit according to any of claims 36-38, wherein the viral infection is an influenza infection.

40. A method of vaccinating a mammal against a viral infection comprising administering the purified IiA protein of any of claims 1-19 in a pharmaceutically acceptable formulation to a human subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

41. A method of vaccinating a mammal against a viral infection comprising administering a purified micelle of claim 20 in a pharmaceutically acceptable formulation to a human subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

42. A method of vaccinating a mammal against a viral infection comprising administering a VLP according to any of claims 21-29 in a pharmaceutically acceptable formulation to a human subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

43. The method according to any of claims 40-42, wherein the pharmaceutically acceptable formulation comprises an adjuvant.

44. The method according to claim 43, wherein the adjuvant is a non-phospholipid liposome,

45. A method of generating an immune response against a viral infection comprising the purified HA protein of any of claims Kl 9 in a pharmaceutically acceptable formulation to a human subject orally, intradermally., intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

46. A method of generating an immune response against a viral infection comprising administering a purified micelle of claim 20 in a pharmaceutically acceptable formulation to a human subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

47. A method of generating an immune response against a viral infection comprising administering a VLP according to any of claims 21-29 in a pharmaceutically acceptable formulation to a human subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

48. A method evaluating an immune response in a vaccine potency assay using a purified HA protein according to any of claims 1-19.

49. The method of claim 48, wherein the vaccine potency assay is a single radial immunodiffusion (SRID) assay.

50. The method of claim 48, wherein the vaccine potency assay is an immunoassay selected from the group consisting of radioimmunoassay, enzyme immunoassay, fluorescence polarization immunoassay (FPlA), Kinetic Interaction of Microparticies in solution (KIMS), lateral flow technology, or T-ceil responses assay.

51. The use of claim 33, wherein the enzyme immunoassay is selected from the group consisting of EMIT, Cloned Enzyme Donor Immunoassay (CEDI), and Enzyme-Linked Immunosorbent Assay (ELISA).

52. A method of purifying a hemagglutinin (HA) protein, comprising steps of:

(i) Expressing said HA in a host cell;

(ii) Extracting said HA from said host cell, wherein said HA is soluble in an extraction buffer;

(iii) Purifying said HA from an extraction buffer using ion exchange chromatography, wherein a pool of tractions containing said HA is produced;

(iv) Purifying said HA from said pool in step (iii) using affinity chromatography, wherein a second pool of fractions containing said HA is produced:

(v) Desalting said second pool in step (iv);

(vi) Purifying said MA from the desalted pool of step (v) using hydroxyapatite chromatography, wherein a third pool of fractions containing said HA is produced; and optionally

(vii) Concentrating, dialysάng, and sterilizing said third pool of fractions containing HA.

53. The method of claim 52, wherein the ion exchange chromatography in step (iii) is an anion exchange chromatography.

54. The method of claim 53, wherein the anion exchange chromatography is a TMAE anion exchange chromatography.

55. The method of any of claims 52-54, wherein the affinity chromatography in step (iv) is a lentil lectin affinity chromatography.

56. The method of any of claims 52-55, wherein the desalting step (v) is conducted with desalting chromatography.

57. The method of claim 56, wherein desalting chromatography is performed using a desalting column packed with Sephadex™ G-25.

58. The method of any of claims 52-57, wherein the desalting step (v) is conducted with dialysis.

59. The method of claim 58, wherein the dialysis is performed using a dialysis bag, tubing, or a stirred celt comprising a selectively permeable membrane.

60. The method of claim 59, wherein the selectively permeable membrane has a N4WCO of about 20 to about 30 kD.

61. The method of any of claims 52-60, wherein the hydroxyapatite chromatography in step (vi) is performed using a column packed with synthetic hydroxyapatite.

62. The method of claim 61, wherein the synthetic hydroxyapatite is ceramic hydroxyapatite.

63. The method of any of claims 52-62, wherein the HA is concentrated and dialyzed in a stirred cell with selectively permeable membrane in step (vii).

64. The method of any of claims 52-63, wherein the HA protein is selected from the group consisting of Hl , H2, H3, H4, H5, H6, H7, H8, H9, HlO, HI l, H12, H13, H14, H15, and Hlό.

65. The method of any of claims 52-64, wherein the HA protein is derived from an influenza virus strain selected from the group consisting of avian influenza virus strain and a mammalian influenza strain.

66. The method of claim 65, wherein the HA is derived from an influenza H lNI virus strain.

67. The method of claim 66, wherein the HlNl virus strain is selected from the group of A/California/04/09, A/New Caledonia/20/ 1999, A/Solomon Is/3/2006, and A/Brisbane/59/2007.

68. The method of any of claims 52-67, wherein said host cell is a eukaryotic cell.

69. The method according to claim 68, wherein said eukaryotic cell is an insect cell.

70. The method according to claim 69, wherein said insect cell is an Sf9 cell.

71. A purified HA protein, wherein the purified HA protein exhibits enhanced or improved antigenicity in a single radial immunodiffusion (SRlD) assay as compared to partially denatured or denatured HA proteins.

72. The purified HA according to any of claims 1-19, wherein the purified HA protein exhibits enhanced or improved antigenicity in a single radial immunodiffusion (SRlD) assay as compared to partially denatured or denatured HA proteins.