HK1194099A - Functional influenza virus like particles (vlps) - Google Patents

Description

Functional influenza virus-like particles (VLPs)

The application is a divisional application of an invention with the application date of 2006, 10, 18, and the Chinese application number of 200680045850.7, and the name of the invention is 'functional influenza virus-like particle (VLP'). .

This application claims priority from the following applications: provisional application 60/727,513 filed on 18/10/2005; provisional application 60/780,847 filed on 10.5.2006; provisional application 60/800,006 filed on 2006, 5, 15; provisional application 60/831,196 filed on 17.7.2006; provisional application 60/832,116 filed on 21/7/2006; and provisional application 60/845,495 filed on 19/9/2006; the entire contents of the above-identified application are hereby incorporated by reference for all purposes.

Background

Influenza viruses are members of the Orthomyxoviridae family (Orthomyxoviridae) (reviewed in Murphy and webster, 1996). There are three subtypes of influenza virus, designated A, B and C. Influenza virions contain a segmented negative sense RNA genome. Influenza virions include the following proteins: hemagglutinin (HA), Neuraminidase (NA), matrix (M1), proton ion channel protein (M2), Nucleoprotein (NP), polymerase basic protein 1(PB1), polymerase basic protein 2(PB2), polymerase acid Protein (PA), and nonstructural protein 2(NS2) proteins. HA. NA, M1 and M2 are membrane-associated, while NP, PB1, PB2, PA and NS2 are nucleocapsid-associated proteins. NS1 is the only non-structural protein that is not bound to virion particles but is specific for influenza virus infected cells. The M1 protein is the most abundant protein in influenza virus particles. The HA and NA proteins are envelope glycoproteins, responsible for viral attachment and entry of viral particles into cells, and are the source of the major immunodominant epitopes used for virus neutralization and protective immunity. Both HA and NA proteins are considered to be the most important components of a prophylactic influenza vaccine.

Influenza virus infection is initiated by attachment of the virion surface HA protein to sialic acid-containing cellular receptors (glycoproteins and glycolipids). NA protein mediates processing of sialic acid receptors, and cellular entry of viruses is dependent on HA-dependent receptor-mediated endocytosis. In the acidic context of internalized endosomes containing influenza virions, the HA protein undergoes a conformational change resulting in fusion of the viral and host cell membranes, followed by viral uncoating, and release of the M1 protein from the nucleocapsid associated Ribonucleoprotein (RNP) mediated by M2 into the nucleus for viral RNA synthesis. Antibodies directed against HA molecules can prevent viral infection by neutralizing viral infectivity, while antibodies directed against NA proteins mediate their effect on the early steps of viral replication.

Currently licensed inactivated influenza a and b vaccines are as trivalent vaccines for parenteral administration. These trivalent vaccines are produced as a monovalent total material in the allantoic cavity of embryonated chicken eggs, purified by rate zonal centrifugation or column chromatography, inactivated with formalin or beta-propiolactone, and formulated into a mixture of two influenza a and b strains prevalent among populations of a particular year. The commercial influenza vaccines available are Whole Virus (WV) or subviral (SV, split or purified surface antigen) virus vaccines. The WV vaccine contains intact inactivated virions. SV vaccines (Flu-Shield, Wyeth-Lederle) treated with solvents such as tri-n-butyl phosphate contain almost all of the viral structural proteins and some of the viral envelopes. The SV vaccine (Fluzone, Sanofi-Aventis; Fluvirin, Novartis) solubilized with Triton X-100 contained mainly aggregates of HA monomers, NA and NP, despite the presence of residual amounts of other viral structural proteins. The FDA recently granted marketing approval for a live attenuated cold-adapted virus vaccine (FluMist, medimmunee) approved for commercial use as an intranasally delivered vaccine for the automatic immunization and prevention of diseases caused by influenza a and b viruses in healthy children 5-17 years of age and young and healthy adults 18-49 years of age.

Several recombinant products have been developed as candidate recombinant influenza vaccines. These approaches all focus on the expression, production and purification of influenza A virus HA and NA proteins, including the expression of these proteins using baculovirus-infected insect cells (Crawford et al,1999; Johansson,1999; Treanor et al, 1996), viral vectors (Pushko et al, 1997; Berglund et al, 1999), and DNA vaccine constructs (Olsen et al, 1997).

Crawford et al (1999) demonstrated that influenza HA expressed in baculovirus-infected insect cells was able to prevent lethal influenza disease caused by H5 and H7 avian influenza subtypes. At the same time, another group demonstrated that baculovirus-expressed influenza HA and NA proteins induced an immune response in animals superior to that induced by one conventional vaccine (Johansson et al, 1999). The immunogenicity and potency of baculovirus-expressed equine influenza virus hemagglutinin were compared to a homologous DNA vaccine candidate (Olsen et al, 1997). In summary, these data demonstrate that high protection against influenza virus challenge can be induced with recombinant HA or NA proteins in different animal models using various experimental approaches.

Lakey et al (1996) showed that influenza HA vaccines derived from baculovirus were well tolerated and immunogenic in human volunteers in a phase I dose escalation safety study. However, in a second phase of the study in which human volunteers were vaccinated at several clinical sites with several doses of influenza vaccine consisting of HA and/or NA proteins, the results showed that the recombinant subunit Protein vaccine did not elicit protective immunity [ G.Smith, Protein Sciences; M.Perdie, USDA, Personal Communications ]. These results indicate that the conformational epitopes displayed on the surface of the infectious virions HA and NA envelope particles (peplomers) are important in the priming of neutralizing antibodies and protective immunity.

Several studies have been conducted on the problem of including other influenza proteins in recombinant influenza vaccine candidates, including experiments involving the influenza nucleoprotein NP (alone or in combination with the M1 protein) (Ulmer et al, 1993; Ulmer et al, 1998; Zhou et al, 1995; Tsui et al, 1998). These vaccine candidates, consisting of approximately invariant internal virion proteins, elicit a broad spectrum of immunity, primarily cellular immunity (CD 4)⁺And CD8⁺Memory T cells). These experiments involve the use of DNA or viral gene vectors. Relatively large amounts of injected DNA are required because experimental results using lower doses of DNA show little or no protection (Chen et al, 1998). Therefore, further preclinical and clinical studies are needed to evaluate whether such DNA-based approaches involving influenza NP and M1 are safe, effective, and durable.

Recently, in an attempt to develop a more effective influenza vaccine, a granule protein was used as a carrier of an epitope of influenza M2 protein. The rationale for developing a vaccine based on M2 was that M2 protein elicited protective immunity against influenza in animal studies (Slepushkin et al, 1995). Neirynck et al (1999) used a 23 amino acid long M2 transmembrane domain as an amino-terminal fusion partner with the hepatitis B virus core antigen (HBcAg) to expose the M2 epitope on the surface of HBcAg capsid-like particles. However, although both the full-length M2 protein and the M2-HBcAg VLP induce detectable antibodies and protection in mice, future influenza vaccines are unlikely to be based solely on the M2 protein because the M2 protein is present in low copy numbers per virion, is poorly antigenic, is unable to elicit antibodies that bind to free influenza virions, and is unable to block viral attachment to cellular receptors (i.e., neutralize the virus).

Since previous studies have shown that the surface influenza glycoproteins HA and NA are the primary targets for eliciting protective immunity against influenza virus, and that M1 provides a conserved target for immunity against influenza cells, new vaccine candidates are likely to comprise these viral antigens as protein macromolecular particles, such as virus-like particles (VLPs). Furthermore, particles with these influenza antigens may display conformational epitopes that can elicit neutralizing antibodies against multiple influenza virus strains.

Several studies have demonstrated that recombinant influenza proteins are capable of self-assembling into VLPs in cell culture using mammalian expression plasmids or baculovirus vectors (Gomez-Puertas et al,1999; Neumann et al, 2000; Latham and Galarza, 2001). Gomez-Puertas et al (1999) demonstrated that efficient formation of influenza VLPs is dependent on the expression levels of viral proteins. Neumann et al (2000) established a mammalian expression plasmid-based system for the generation of infectious influenza virus-like particles entirely from cloned cDNA. Latham and Galarza (2001) reported the formation of influenza VLPs in recombinant baculovirus-infected insect cells that co-express the HA, NA, M1 and M2 genes. These studies demonstrate that influenza virion proteins self-assemble when co-expressed in eukaryotic cells.

Summary of The Invention

The present invention provides a virus-like particle (VLP) comprising the influenza virus M1 protein and the influenza viruses H5 and N1 hemagglutinin and neuraminidase proteins. In one embodiment, the M1 protein is derived from a different influenza strain than the H5 and N1 proteins. In another embodiment, the H5 or N1 is from an H5N1 clade (clade)1 influenza virus.

The invention also provides VLPs expressed by eukaryotic cells comprising one or more nucleic acids encoding influenza H5 and N1 proteins and influenza M1 protein under conditions that allow VLPs to be formed. In one embodiment, the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, an amphibian cell, an avian cell, and a mammalian cell. In another embodiment, the eukaryotic cell is an insect cell.

The invention also provides a VLP which, when administered to a human or animal, elicits neutralizing antibodies in the human or animal body that are protective against influenza infection.

The invention also provides an immunogenic composition comprising an effective dose of a VLP of the invention. In one embodiment, the composition comprises an adjuvant.

The invention also provides a vaccine comprising an effective dose of a VLP of the invention. In one embodiment, the vaccine comprises at least a second VLP comprising HA and NA from different influenza virus strains. In another embodiment, the vaccine comprises an adjuvant.

The invention also provides a method of inducing significant immunity against influenza virus infection in an animal comprising administering at least one effective dose of a vaccine comprising a VLP of the invention. In one embodiment, the vaccine is administered to the animal orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

The invention also provides the use of the VLP of the invention for the preparation of a vaccine for an animal, wherein the vaccine induces significant immunity against infection by influenza virus in said animal.

The invention also provides a method of making the VLP of the invention comprising expressing M1, HA and NA proteins in a eukaryotic cell.

The present invention provides a vaccine comprising an influenza VLP, wherein said VLP comprises influenza M1, HA and NA proteins, wherein said vaccine induces significant immunity in humans against influenza virus infection. In one embodiment, the vaccine comprises an influenza VLP, wherein the VLP consists essentially of influenza M1, HA and NA proteins, wherein the vaccine induces significant immunity in humans against influenza virus infection. In another embodiment, the vaccine comprises an influenza VLP, wherein the VLP comprises a plurality of influenza proteins selected from the group consisting of influenza M1, HA and NA proteins, wherein the vaccine induces significant immunity against influenza virus infection in humans.

The invention also provides the use of an influenza VLP, wherein said VLP comprises influenza M1, HA and NA proteins, for the preparation of a vaccine, wherein said vaccine induces significant immunity in humans against influenza virus infection.

Thus, the present invention provides a macromolecular protein structure comprising (a) a first influenza virus M1 protein and (b) an additional structural protein, which may include: a second or more influenza virus M1 protein; a first, second or more influenza virus HA protein; a first, second or more influenza virus NA protein; and a first, second or more influenza virus M2 protein. If the additional structural protein is not from the second or more influenza M1 virus proteins, then all members of the group, such as the first and second influenza M2 virus proteins, are included. Thus, the present invention provides a functional influenza protein structure consisting essentially of the influenza virus structural proteins produced by the methods of the invention, including subviral particles, VLPs or capsomere structures, or portions thereof, vaccines, multivalent vaccines and mixtures thereof. In a particularly preferred embodiment, the influenza macromolecular protein structure comprises influenza HA, NA and M1 proteins that are the expression products of influenza genes cloned as synthetic fragments from wild-type viruses.

The macromolecular protein structure may also include additional structural proteins such as Nucleoprotein (NP), membrane proteins from species other than non-influenza viruses (non-influenza viruses) and membrane proteins from non-influenza sources (non-influenza sources) derived from avian or mammalian sources and different subtypes of influenza viruses, including influenza a and b viruses. The present invention may include chimeric macromolecular protein structures comprising at least one protein having a moiety that is not produced by influenza virus.

Prevention of influenza can be achieved by providing macromolecular protein structures that can self-assemble from recombinant constructs in host cells. The macromolecular protein structures of the present invention are capable of self-assembling into homotypic or heterotypic virus-like particles (VLPs) that display conformational epitopes on the HA and NA proteins, which epitopes elicit protective neutralizing antibodies. The composition may be a vaccine composition which further comprises a carrier or diluent and/or an adjuvant. Functional influenza VLPs are capable of eliciting neutralizing antibodies against one or more influenza virus strains or virus types, depending on whether the functional influenza VLPs contain HA and/or NA proteins from one or more virus strains or virus types. The influenza virus protein comprised by the vaccine may be a wild-type influenza virus protein. Preferably, the structural proteins, or parts thereof, comprising the influenza VLPs may be derived from multiple strains of wild-type influenza virus. The influenza vaccine may be administered to a human or animal to elicit protective immunity against one or more influenza virus strains or virus types.

The macromolecular protein structures of the present invention may exhibit hemagglutinin activity and/or neuraminidase activity.

The present invention provides a method of making influenza-derived VLPs by constructing a recombinant construct that encodes an influenza structural gene (including M1, HA) and at least one influenza virus-derived structural protein. Using the recombinant construct, a suitable host cell is transfected, infected or transformed with a recombinant baculovirus. Culturing the host cell under conditions that allow expression of M1, HA and at least one influenza virus-derived structural protein, forming VLPs in the host cell. Culture broth of infected cells containing functional influenza VLPs was harvested and VLPs were purified. Yet another feature of the invention is the additional step of co-transfecting, co-infecting or co-transforming the host cell with a second recombinant construct encoding a second influenza protein, thereby incorporating the second influenza protein into the VLP. Such structural proteins may be derived from influenza viruses, including NA, M2 and NP, and at least one structural protein is derived from avian or mammalian sources. The structural proteins may be influenza a and b viruses. According to the invention, the host cell may be a eukaryotic cell. Furthermore, the VLP may be a chimeric VLP.

Another feature of the invention is a method of formulating a pharmaceutical product containing influenza VLPs by introducing into a host cell a recombinant construct encoding an influenza virus gene and allowing the recombinant influenza virus protein to self-assemble into functional homo-or heterotypic VLPs in the cell. Isolating and purifying influenza VLPs, and formulating a pharmaceutical product containing the influenza VLPs. The pharmaceutical product may further comprise an adjuvant. Furthermore, the present invention provides a method of formulating a drug product by mixing such a drug product containing influenza VLPs with lipid vesicles, i.e. non-ionic lipid vesicles. Thus, functional homo-or hetero-VLPs can bud (bud) from infected cells as enveloped particles. The budding influenza VLPs can be isolated and purified as pharmaceuticals by ultracentrifugation or column chromatography, either alone or with adjuvants such as(Novavax, product Inc.) and the like into pharmaceutical products, such as vaccines.Providing an enhanced immune effect, further described in U.S. patent No.4,911,928, incorporated herein by reference.

The present invention provides a method of detecting humoral immunity to influenza virus infection in a vertebrate by providing a test agent comprising an effective antibody detecting amount of an influenza virus protein having at least one conformational epitope of an influenza virus macromolecular structure. The test agent is contacted with a sample of bodily fluid from a vertebrate in need of examination for influenza virus infection. The influenza virus-specific antibody contained in the sample is allowed to bind to a conformational epitope of the influenza virus macromolecular structure to form an antigen-antibody complex. These complexes are separated from unbound complexes and contacted with a detectably labeled immunoglobulin binding agent. Determining the amount of detectably labeled immunoglobulin binding agent bound to the complex.

Influenza viruses can be detected in a specimen from an animal or human suspected of being infected with the influenza virus by providing an antibody having a label that produces a detectable signal or linked to a detectably labeled reagent and specific for at least one conformational epitope of the influenza virus particle. The specimen is contacted with the antibody and the antibody is allowed to bind to the influenza virus. Determining the presence of influenza virus in the specimen using the detectable label.

The present invention provides methods of treating, preventing and generating a protective immune response by administering to a vertebrate an effective amount of a composition of the invention.

Alternatively, the influenza VLP drug may be formulated into a laboratory reagent for structural studies of influenza virus and clinical diagnostic assays. The invention also provides kits and instructions for use in treating influenza virus by administering an effective amount of the compositions of the invention.

The invention also provides VLPs comprising HA, NA and M1 proteins derived from avian influenza virus capable of causing morbidity (morbidity) or mortality (mortality) in vertebrates. In one embodiment, the HA, NA and M1 proteins are derived from avian influenza a virus. In another embodiment, HA is selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16, and NA is selected from N1, N2, N3, N4, N5, N6, N7, N8 and N9. In another embodiment, the HA and NA proteins are H5 and N1, respectively. In another embodiment, the HA and NA proteins are H9 and N2, respectively. In another embodiment, the HA and/or NA exhibit hemagglutinin activity and/or neuraminidase activity, respectively. In one embodiment, the VLP consists essentially of HA, NA and M1 proteins, that is, these proteins are essentially the only influenza proteins in the VLP.

The invention also provides a method of producing a VLP comprising transfecting a vector encoding an avian influenza virus protein into a suitable host cell and expressing the avian influenza virus protein under conditions that allow formation of a VLP. In one embodiment, the method involves transfecting a host cell with a recombinant DNA molecule encoding only HA, NA, and M1 influenza proteins.

The invention also includes an antigenic preparation comprising VLPs containing HA, NA and M1 proteins derived from avian influenza virus capable of causing morbidity and mortality in vertebrates. In another embodiment, HA is selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16, and NA is selected from N1, N2, N3, N4, N5, N6, N7, N8 and N9. In another embodiment, the HA and NA proteins are H5 and N1, respectively. In another embodiment, the HA and NA proteins are H9 and N2, respectively. In another embodiment, the antigenic preparation is administered to the subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

The invention further provides a method of immunizing a vertebrate against avian influenza virus, comprising administering to the vertebrate an inducibly protective amount of VLPs comprising HA, NA and M1 proteins derived from avian influenza virus.

The invention also includes a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of influenza VLPs. In one embodiment, the VLP consists essentially of HA, NA, and M1. In another embodiment, the VLP comprises an influenza protein, wherein the influenza protein consists of HA, NA, and M1. In another embodiment, the HA and/or NA exhibit hemagglutinin and/or neuraminidase activity, respectively.

The invention also includes a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of avian influenza VLPs. In one embodiment, the influenza VLP consists essentially of avian HA, NA and M1. In another embodiment, the influenza VLP comprises an influenza protein, wherein the influenza protein consists of avian HA, NA, and M1.

The invention also includes a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of seasonal influenza VLPs. In one embodiment, the influenza VLP consists essentially of seasonal HA, NA, and M1. In another embodiment, the influenza VLPs comprise influenza proteins, wherein the influenza proteins consist of seasonal HA, NA, and M1.

The invention also includes a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of at least one seasonal influenza VLP. In one embodiment, the influenza VLPs comprise seasonal influenza HA, NA, and M1. In another embodiment, the influenza VLP consists essentially of seasonal influenza HA, NA, and M1.

The invention further includes a method of inducing a significant protective antibody response against an influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of influenza VLPs.

The invention includes a method of inducing a significant protective cellular immune response against influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of influenza VLPs.

The invention also includes a method of formulating a vaccine that induces significant immunity in a subject against influenza virus infection or at least one symptom thereof, comprising adding an effective dose of influenza VLPs to the formulation. In one embodiment, the significant immunity to influenza virus infection or at least one symptom thereof is conferred by a dose. In another embodiment, the significant immunity to influenza virus infection or at least one symptom thereof is conferred by multiple doses.

The invention also includes a vaccine comprising influenza VLPs, wherein the vaccine induces significant immunity against influenza virus infection or at least one symptom thereof when administered to a subject. In one embodiment, the influenza VLPs are avian influenza VLPs. In another embodiment, the influenza VLPs are seasonal influenza VLPs.

The invention also includes an antigenic preparation comprising influenza VLPs, wherein said vaccine induces significant immunity against influenza virus infection or at least one symptom thereof when administered to a subject. In one embodiment, the influenza VLPs are avian influenza VLPs. In another embodiment, the influenza VLPs are seasonal influenza VLPs.

Brief description of the drawings

FIG. 1 depicts the nucleotide sequence (SEQ ID NO:1) of the Neuraminidase (NA) gene of the avian influenza A/hong Kong/1073/99 (H9N2) virus.

FIG. 2 depicts the nucleotide sequence (SEQ ID NO:2) of the avian influenza A/hong Kong/1073/99 (H9N2) virus Hemagglutinin (HA) gene.

FIG. 3 depicts the nucleotide sequence (SEQ ID NO:3) of the avian influenza A/hong Kong/1073/99 (H9N2) virus matrix protein M1(M1) gene.

FIG. 4 depicts the construction of a transfer vector for recombinant baculovirus expressing avian influenza A/hong Kong/1073/99 (H9N2) HA, NA and M1 proteins. Fig. 4 (a) depicts a transfer vector expressing each gene, and fig. 4 (B) depicts a transfer vector for multiple expression of genes.

FIG. 5 depicts the expression of avian influenza A/hong Kong/1073/99 (H9N2) HA, NA and M1 proteins in Sf-9S cells.

Figure 6 depicts purification of avian influenza a/hong kong/1073/99 (H9N2) VLPs by sucrose density gradient method.

FIG. 7 depicts detection of influenza virus proteins by gel filtration chromatography. The antibodies used in the Western blot analysis were as follows: (A) rabbit anti-H9N 2; (b) murine anti-M1 mab; (C) mouse anti-BACgp 64.

Figure 8 depicts detection of avian influenza a/hong kong/1073/99 (H9N2) proteins, including subviral particles, VLPs and VLP complexes, by electron microscopy.

Fig. 9 depicts hemagglutinin activity of purified avian influenza a/hong kong/1073/99 (H9N2) VLPs.

Figure 10 depicts neuraminidase activity of purified avian influenza a/hong kong/1073/99 (H9N2) VLPs.

Figure 11 depicts immunization and bleeding schedules used for recombinant influenza immunogenicity studies in mice using purified avian influenza a/hong kong/1073/99 (H9N2) VLPs.

Figure 12 depicts the results of immunogenicity studies in mice immunized with recombinant influenza H9N2 VLPs. Figure 12 (a) depicts serum from BALB/c mice immunized with recombinant VLPs consisting of HA, NA and M1 proteins from avian influenza a/hong kong/1073/99 (H9N 2). FIG. 12 (b) depicts a Western blot reaction of sera from New Zealand white rabbits immunized with inactivated avian influenza A virus H9N2 with either inactivated avian influenza A virus H9N2 (lanes 1 and 3) or cold-adapted avian influenza A virus H9N2 (lanes 2 and 4).

FIG. 13 depicts the geometric mean antibody response in BALB/c mice after primary and secondary immunizations.

FIG. 14 depicts serum Hemagglutinin Inhibition (HI) responses in BALB/c mice.

FIG. 15 depicts weight loss (%) in BALB/c mice challenged with H9N2 influenza.

Figure 16 depicts pneumovirus titers 3 and 5 days post challenge with H9N 2.

Fig. 17A, 17B and 17C depict antibody responses against type a/fujian/411/2002 following immunization of mice with H3N2 VLPs.

Fig. 18A and 18B depict mouse IgG antibody isotypes.

Figure 19 depicts Hemagglutinin Inhibition (HI) antibody responses in SD rats immunized with the H9N2VLP vaccine.

FIGS. 20A and 20B depict Hemagglutinin Inhibition (HI) antibody responses against different doses of H9N2 VLPs with and without adjuvant in BALB/c mice.

FIG. 21 depicts serum Hemagglutinin Inhibition (HI) responses between different doses of VLPs in BALB/c mice.

Figure 22 depicts serum Hemagglutinin Inhibition (HI) responses in ferrets.

FIG. 23 depicts serum Hemagglutinin Inhibition (HI) responses of sera drawn at 21 and 42 days after ferrets were administered H3N2 VLPs of different virus strains.

Figure 24 depicts anti-HA antibodies (end-point dilution titers) of mice vaccinated intramuscularly with low dose H5N1 (vietnam/1203/2003) VLPs.

FIG. 25 depicts anti-HA antibodies (end-point dilution titers) of mice intranasally vaccinated with low-dose H5N1 (Vietnam/1203/2003) VLPs.

Fig. 26 depicts one example of the manufacture, isolation and purification of VLPs of the invention.

FIG. 27 depicts mice vaccinated by intramuscular administration of H3N2 VLPs, followed by intranasal challenge with A/Aichi/2/68 x31(H3N2) virus.

FIG. 28 depicts mice vaccinated by intranasal administration of H3N2 VLPs and then challenged intranasally with A/Aichi/2/68 x31(H3N2) virus.

Figure 29 depicts viral shedding (shedding) in nasal wash of ferrets following vaccination with H9N2VLP and intranasal challenge with H9N2 virus.

Figures 30A, 30B, 30C, 30D, 30E, 30F, 30G and 30H depict Hemagglutinin Inhibition (HI) antibody responses in mice tested against different influenza virus H3N2 strains following intramuscular or intranasal vaccination with different doses of a/fujian/411/2002 (H3N2) VLPs.

Detailed Description

As used herein, the term "baculovirus", also known as baculoviridae (baculoviridae), refers to a family of arthropod enveloped DNA viruses, members of which can be used as expression vectors to produce recombinant proteins in insect cell culture. Virosomes comprise one or more rod-shaped nucleocapsids, together with circular supercoiled double-stranded DNA molecules (Mr 54X 10)⁶-154×10⁶). The virus used as a vector is usually Autographa californica nuclear polyhedrosis virus (NVP). Expression of the introduced gene is under the control of a strong promoter that normally regulates the polyhedrin component of large nuclear inclusion bodies in which the virus is embedded in infected cells.

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

As used herein, the term "first" influenza virus protein, i.e., the first influenza virus M1 protein, refers to proteins derived from a particular influenza virus strain, e.g., M1, HA, NA, and M2. The strain or type of the first influenza virus is different from the strain or type of the second influenza virus protein. Thus, a "second" influenza virus protein, i.e., a second influenza virus M1 protein, refers to a protein derived from a second influenza virus strain, e.g., M1, HA, NA, and M2, wherein the second influenza virus strain is a different strain or type than the first influenza virus protein.

As used herein, the term "hemagglutinin activity" refers to the ability of an HA-containing protein, VLP or portion thereof to bind to and agglutinate red blood cells (erythrocytes).

As used herein, the term "neuraminidase activity" refers to the enzymatic activity possessed by NA-containing proteins, VLPs or portions thereof to cleave sialic acid residues from a substrate (including proteins such as fetuin).

As used herein, the term "heterotypic" (heterologous) refers to one or more different types or strains of virus.

As used herein, the term "homotypic" (heterotypic) refers to a type or strain of virus.

As used herein, the term "macromolecular protein structure" refers to the structure (conformation) or arrangement (arrangement) of one or more proteins.

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

As used herein, the term "non-influenza" (non-influenza) refers to a protein or molecule that is not derived from an influenza virus.

As used herein, the term "vaccine" refers to a preparation of a killed or weakened pathogen, or a preparation of derived antigenic determinants, for inducing the formation of antibodies or immunity against said pathogen. Vaccines are administered to provide immunity against disease, such as influenza caused by influenza virus. The present invention provides vaccine compositions that are immunogenic and provide protection. In addition, the term "vaccine" also refers to a suspension or solution of an immunogen (e.g., VLP) that is administered to a vertebrate to generate protective immunity, i.e., immunity that reduces the severity of infection-related disease.

As used herein, the term "significant immunity" refers to an immune response that is: when the VLP of the present invention is administered to a vertebrate, there is induction of the immune system in the vertebrate, resulting in prevention of influenza infection, amelioration of influenza infection, or reduction of at least one symptom associated with influenza virus infection in the vertebrate. Significant immunity may also refer to a Hemagglutination Inhibition (HI) titer of > 40 in a mammal to which the VLPs of the invention have been administered and an immune response has been induced.

As used herein, the term "adjuvant" refers to a compound that, when used in combination with a particular immunogen (e.g., VLP) in a formulation, increases or otherwise alters or modifies the immune response resulting therefrom. Modification of the immune response includes an increase in the intensity or an increase in specificity of one or both of the antibody immune response and the cellular immune response. Modification of the immune response may also mean a reduction or suppression of a particular antigen-specific immune response.

As used herein, the term "immunostimulant" refers to a compound that enhances the immune response through the body's own chemical messengers (cytokines). These molecules include various cytokines, lymphokines, and chemokines, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13) with immunostimulatory, immunopotentiating, and proinflammatory activities; growth factors (e.g., granulocyte-macrophage (GM) Colony Stimulating Factor (CSF)); and other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 ligand, b.71; b7.2 and the like. The immunostimulatory molecule may be administered in the same formulation as the influenza VLP, or may be administered separately. The protein or expression vector encoding the protein may be administered to produce an immunostimulatory effect.

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

As used herein, the term "avian influenza virus" refers to an influenza virus that is found primarily in birds, but may also infect humans or other animals. In some cases, avian influenza viruses can spread or spread from one person to another. Avian influenza viruses that infect humans have the potential to cause influenza pandemics, i.e., morbidity and mortality in humans. A pandemic occurs when a new strain of influenza virus (a virus to which a person does not have natural immunity) appears, which spreads through various sites and possibly throughout the world, while infecting many people.

As used herein, the term "seasonal Influenza virus" refers to an Influenza virus strain that has been determined to be spreading within a human population for a certain Influenza season based on epidemiological investigations by National Influenza Centers around the world. These epidemiological findings, as well as certain isolated influenza viruses, were sent to one of four reference laboratories of the World Health Organization (WHO) for detailed study, one of which was located at the Centers for disease Control and Prevention (CDC) in atlanta. These laboratories tested how well antibodies prepared against existing vaccines reacted with circulating viruses and new influenza viruses. This information and information about flu activity were aggregated and submitted to an advisory board of the U.S. Food and Drug Administration (FDA) and at the WHO's conference. The result of these conferences was the selection of three viruses (two subtypes of influenza a virus and one subtype of influenza b virus) into an influenza vaccine for the next fall and winter. The selection was made in the northern hemisphere in month 2 and in the southern hemisphere in month 9. Typically, one or two of the three strains in a vaccine are changed annually.

As used herein, the term "significantly protective antibody response" refers to an immune response exhibited by a vertebrate (e.g., a human) that is mediated by antibodies to influenza virus, prevents or ameliorates influenza infection, or reduces at least one symptom thereof. The VLPs of the invention may stimulate the production of antibodies (e.g., neutralizing antibodies) that prevent influenza virus from entering the cell, bind to the influenza virus thereby blocking the virus replication, and/or protect host cells from infection and destruction.

As used herein, the term "significantly protective cellular immunity" refers to an immune response exhibited by a vertebrate (e.g., a human) that is mediated by T lymphocytes and/or other leukocytes against influenza virus, which prevents or ameliorates influenza infection or reduces at least one symptom thereof. An important aspect of cellular immunity relates to the antigen-specific response by cytolytic T Cells (CTLs). CTLs are specific for peptide antigens that are presented in association with Major Histocompatibility Complex (MHC) -encoded proteins and expressed on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms or the lysis of cells infected with these microorganisms. Another aspect of cellular immunity relates to antigen-specific responses by T helper cells. T helper cells help stimulate the function of non-specific effector cells and focus the activity of these effector cells towards cells displaying on their surface peptide antigens bound to MHC molecules. "cellular immune response" also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and/or other leukocytes, including cells derived from CD4+ and CD8+ T cells.

As used herein, the term "significant immunity based on the population at large" refers to the immunity caused by administration of the VLP of the invention to individuals in the population. Immunity in the individual in the population results in prevention, amelioration of influenza infection in the individual, or reduction of at least one symptom associated with influenza virus infection, and prevents spread of the influenza virus to other individuals in the population. The term "population" is defined as a group of individuals (e.g., schoolchildren, elderly people, healthy individuals, etc.) that may include a geographic area (e.g., a particular city, school, cell, workplace, country (county), state (state), etc.).

As used herein, the term "antigenic preparation" or "antigenic composition" refers to a preparation that: when administered to a vertebrate, particularly an avian or mammalian species, an immune response is induced.

As used herein, the term "vertebrate" or "subject" or "patient" refers to any member of the subphylum chordata (suphylum cordiata), including but not limited to humans and other primates, including non-human primates, such as chimpanzees and other apes and monkey species. Livestock (farm animals), such as cattle, sheep, pigs, goats, and horses; domestic animals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including poultry, wild and game birds, such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like are also non-limiting examples. The terms "mammal" and "animal" are included in this definition. Intended to cover both adult and newborn individuals.

Although specific inactivated virus vaccines exist that are 60-80% effective under optimal conditions, influenza remains a common public health problem. When these vaccines are effective, the disease is usually avoided by preventing viral infection. Accumulation of antigenic differences (antigenic shift and antigenic drift) can lead to vaccine failure. For example, avian influenza a H9N2 is co-circulating with human influenza virus a/sydney/97 (H3N2) in pigs (co-circulating), leading to gene rearrangement and the emergence of new strains of human influenza virus with pandemic potential (Peiris et al, 2001). In the event of such antigenic shift, existing vaccines are unlikely to provide adequate protection.

Another reason for the lack of influenza vaccine regimens is the relatively short duration of immunity elicited by existing vaccines. Further lack of influenza control measures reflects the limited use of existing vaccines due to vaccine reactogenicity and side effects to young children, the elderly, and people allergic to egg components used in the manufacture of commercially licensed inactivated virus influenza vaccines.

In addition, inactivated influenza virus vaccines often lack or have alterations in the conformational epitopes of HA and NA that are present, and these epitopes elicit neutralizing antibodies and play an important role in immunity against disease. Thus, inactivated virus vaccines and certain recombinant monomeric influenza subunit protein vaccines confer inadequate protection. On the other hand, macromolecular protein structures, such as capsomeres, subviral particles and/or VLPs, containing multiple copies of the native protein displaying conformational epitopes, are advantageous for obtaining optimal vaccine immunogenicity.

The present invention describes the cloning of avian influenza a/hong kong/1073/99 (H9N2) virus HA, NA and M1 genes individually or in tandem into a single baculovirus expression vector, and the production of influenza vaccine candidates or reagents composed of recombinant influenza structural proteins; in baculovirus infected insect cells, these recombinant influenza structural proteins self-assemble into functional and immunogenic homotypic macromolecular protein structures, including subviral influenza particles and influenza VLPs.

The invention describes the cloning of human influenza A/Sydney/5/97 and A/Fujian/411/2002 (H3N2) virus HA, NA, M1, M2 and NP genes into baculovirus expression vectors, and the production of influenza vaccine candidates or reagents composed of influenza structural proteins; in baculovirus infected insect cells, these influenza structural proteins self-assemble into functional and immunogenic homomacromolecular protein structures, including subviral influenza particles and influenza VLPs.

Furthermore, the present invention describes the cloning of the HA gene of human influenza a/sydney/5/97 and a/fujian/411/2002 (H3N2) virus and the HA, NA and M1 genes of avian influenza a/hong kong/1073/99 (H9N2) in tandem into a single baculovirus expression vector, and the production of influenza vaccine candidates or reagents composed of influenza structural proteins; in baculovirus infected insect cells, these influenza structural proteins self-assemble into functional and immunogenic homomacromolecular protein structures, including subviral influenza particles and influenza VLPs.

The VLP of the invention

The influenza VLPs of the present invention are useful for the preparation of vaccines against influenza viruses. An important feature of this system is the ability to replace surface glycoproteins with different subtypes of HA and/or NA or other viral proteins, so that new antigenic variants of influenza can be renewed each year or to prepare for influenza pandemics. When antigenic variants of these glycoproteins are identified, the VLPs can be upgraded to include these new variants (e.g., for use in seasonal influenza vaccines). In addition, surface glycoproteins from potentially pandemic viruses such as H5N1, or other HA, NA combinations with pandemic potential, can also be incorporated into VLPs without fear of releasing genes that have not been prevalent in humans for decades. This is because these VLPs are not infectious, do not replicate and are not pathogenic. Thus, the system can be used to create new candidate influenza vaccines each year, and/or to create influenza pandemic vaccines whenever needed.

There are 16 different Hemagglutinins (HA) and 9 different Neuraminidases (NA), all of which are found in wild birds. Wild birds are the main natural reservoir of all types of influenza a virus and are considered the source of all types of influenza a virus in all other vertebrates. These subtypes differ due to changes in Hemagglutinin (HA) and Neuraminidase (NA) on their surface. Many different combinations of HA and NA proteins are possible. Each combination represents a different influenza a virus type. In addition, each type can be further classified into multiple virus strains according to the different mutations found in each of its 8 genes.

All known types of influenza a viruses can be found in birds. Generally, avian influenza viruses do not infect humans. However, certain avian influenza viruses can develop genetic variations associated with the ability to cross the species barrier. Such viruses can cause pandemics because humans do not have natural immunity to the virus and can readily transmit from person to person. In hong kong 1997, in one outbreak of avian influenza in poultry, avian influenza virus was transmitted from birds to humans. This virus was identified as influenza virus H5N 1. This virus causes a severe respiratory illness in 18 people, of which 6 die. Since then, an increasing number of known cases of H5N1 infection have occurred in humans worldwide, about half of which have died.

Thus, the invention includes cloning HA, NA, and M1 nucleotides into an expression vector from avian influenza virus, influenza virus with pandemic potential, and/or seasonal influenza virus. The invention also describes the production of influenza vaccine candidates or reagents consisting of influenza proteins that self-assemble into functional VLPs. All viral protein combinations must be co-expressed with M1 nucleotide.

The VLPs of the invention comprise or consist of influenza HA, NA and M1 proteins. In one embodiment, the VLP comprises HA from avian influenza virus, pandemic influenza virus and/or seasonal influenza virus, wherein the HA is selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16, and NA from avian influenza virus, pandemic influenza virus and/or seasonal influenza virus selected from N1, N2, N3, N4, N5, N6, N7, N8 and N9. In another embodiment, the invention includes a VLP consisting essentially of HA, NA and M1. The HA and NA may be from the HA and NA listed above. These VLPs may contain negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, the influenza VLP comprises an influenza protein, wherein the influenza protein consists of HA, NA, and M1 proteins. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, HA and/or NA may exhibit hemagglutinin activity and/or neuraminidase activity, respectively, when expressed on the surface of a VLP.

In another embodiment, the VLP comprises HA and NA of H5N1 virus, and M1 protein (which may or may not be from the same virus strain). In another embodiment, the VLP consists essentially of HA and NA of H5N1 virus and M1 protein. These VLPs may contain negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, the VLP consists of HA and NA of H5N1 virus and M1 protein. In another embodiment, the influenza VLPs comprise influenza proteins, wherein the influenza proteins consist of H5, N1, and M1 proteins. These VLPs contain H5, N9 and M1, and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, H5 and/or N1). In another embodiment, H5 and/or N1 may exhibit hemagglutinin activity and/or neuraminidase activity, respectively, when expressed on the surface of a VLP. In another embodiment, the VLP comprises HA and NA of H9N2 virus, and M1 protein. In another embodiment, the VLP consists essentially of HA and NA of H9N2 virus, and M1 protein. These VLPs may contain negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, the VLP consists of HA and NA of H9N2 virus, and M1 protein. In another embodiment, the influenza VLPs comprise influenza proteins, wherein the influenza proteins consist of H9, N2, and M1 proteins. These VLPs contain H9, N2 and M1, and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, H9 and/or N2). In another embodiment, H9 and/or N2 may exhibit hemagglutinin activity and/or neuraminidase activity, respectively, when expressed on the surface of a VLP.

In another embodiment, the VLP comprises HA and NA from influenza b virus, and M1 protein. Influenza b viruses are usually found only in humans. Unlike influenza a viruses, these viruses are not classified according to subtype. Influenza b virus can cause morbidity and mortality in humans, but its associated epidemics are generally of lesser severity compared to influenza a virus. In another embodiment, the VLP consists essentially of HA and NA from influenza b virus, and M1 protein. These VLPs may contain negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, the influenza VLP comprises an influenza protein, wherein the influenza protein consists of HA, NA, and M1 proteins. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, the VLP consists of HA and NA of influenza b virus, and M1 protein. In another embodiment, HA and/or NA may exhibit hemagglutinin activity and/or neuraminidase activity, respectively, when expressed on the surface of a VLP.

The invention also encompasses variants of said influenza protein expressed on or in the VLP of the invention. These variants may comprise alterations in the amino acid sequence of the component protein. The term "variant" with respect to a polypeptide refers to an amino acid sequence that has been altered by one or more amino acids relative to a reference sequence. Variants may have "conservative" changes, where the amino acid used for substitution has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. Alternatively, a variant may have "non-conservative" changes, such as the replacement of glycine with tryptophan. Similar minor changes may also include amino acid deletions and/or insertions. Using computer programs well known in the art, such as DNASTAR software, guidance can be found for determining which amino acid residues are available for substitution, insertion, or deletion without abolishing biological or immunological activity.

Natural variants may arise due to antigenic drift. Antigenic drift is a small change in viral proteins that occurs constantly over time. Thus, a person infected with a particular influenza strain produces antibodies against that virus, and when a new strain appears, antibodies against the older strain no longer recognize the newer virus, and reinfection may occur. This is why there is a new influenza vaccine every season. In addition, certain alterations in influenza virus can result in cross-species spread of influenza virus. For example, certain avian influenza viruses develop genetic variations that are associated with the ability to cross species barriers. Such viruses can cause pandemics because humans do not have natural immunity to the virus, which can be readily transmitted from person to person. Naturally occurring variations of the influenza proteins are an embodiment of the invention.

General textbooks describing molecular biology techniques such as cloning, mutation, cell culture, etc. applicable to the present invention include: berger and Kimmel, Guide to Molecular Cloning technologies, Methods in Enzymology volume152Academic 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. Autosubel et al, materials, Current Protocols, by Greene Publishing Associates, Inc. and n Wiley & Sons, Inc. incorporated. These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics, such as cloning and mutation of HA and/or NA molecules, etc. Thus, the invention also encompasses methods of using known protein engineering and recombinant DNA techniques to improve or alter the properties of influenza proteins expressed on or in the VLPs of the invention. There are a variety of mutagenesis types that can be used to generate and/or isolate variant HA, NA and/or M1 molecules and/or to further modify/mutate the polypeptides of the invention. They include, but are not limited to, site-directed mutagenesis, random site mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, and the like. Other suitable methods include point mismatch repair (point mismatch repair), mutagenesis using repair-deficient host strains, restriction selection and restriction purification, deletion mutagenesis, mutagenesis by whole gene synthesis, double-strand break repair, and the like. Mutagenesis, e.g., involving chimeric constructs, is also encompassed by the present invention. In one embodiment, mutagenesis can be guided by known information of the naturally occurring molecule or naturally occurring molecule that has been altered or mutated, e.g., sequence comparison, physical properties, crystal structure, and the like.

The invention also includes influenza protein variants that exhibit significant biological activity (e.g., are capable of eliciting an effective antibody response) when expressed on or in VLPs. Such variants include deletions, insertions, inversions, duplications, and substitutions, which are selected so as to have little effect on activity according to general rules known in the art.

Methods for cloning the influenza proteins are known in the art. For example, polyadenylated mRNA can be extracted from cells infected with influenza virus, from which influenza genes encoding specific influenza proteins are isolated by RT-PCR. The resulting product gene can be cloned into a vector as a DNA insert. The term "vector" refers to a tool that can be used to amplify nucleic acids and/or transfer nucleic acids between organisms, cells, or cellular components. Vectors include plasmids, viruses, phages, proviruses, phagemids, transposons, artificial chromosomes and the like that replicate autonomously or can integrate into the chromosome of the host cell. The vector may also be a naked RNA polynucleotide that is non-autonomously replicating, a naked DNA polynucleotide, a polynucleotide comprising both DNA and RNA in the same strand, a polylysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, and the like. In many, but not all, typical embodiments, the vectors of the invention are plasmids or bacmid (bacmid).

Thus, the invention includes nucleotides encoding HA, NA and/or M1 influenza proteins cloned into an expression vector capable of expression in a cell, wherein the vector induces the formation of VLPs. An "expression vector" is a vector, such as a plasmid, capable of promoting expression and replication of a nucleic acid incorporated therein. Typically, the nucleic acid to be expressed is "operably linked" to and under the transcriptional control of a promoter and/or enhancer. In one embodiment, the nucleotide encoding HA from avian influenza virus, pandemic influenza virus and/or seasonal influenza virus is selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In another embodiment, the nucleotides encoding NA from avian influenza virus, pandemic influenza virus and/or seasonal influenza virus are selected from N1, N2, N3, N4, N5, N6, N7, N8 and N9. In another embodiment, the vector consists of nucleotides encoding HA, NA and/or M1 influenza proteins. In another embodiment, the vector consists of nucleotides encoding HA, NA and M1 influenza proteins. Preferred expression vectors are baculovirus vectors. After cloning the nucleotides encoding the influenza proteins, the nucleotides can be further manipulated. For example, one skilled in the art can mutate a particular base of a coding region to create a variant. Variants may contain alterations in coding regions, non-coding regions, or both coding and non-coding regions. Such variants may increase the immunogenicity of the influenza protein or remove splice sites in the protein or RNA. For example, in one embodiment, the donor and acceptor splice sites on the influenza M protein (full length) are mutated to avoid splicing of M mRNA into M1 and M2 transcripts. In another embodiment, HA is engineered to remove or mutate the cleavage site. For example, wild-type H5HA has a cleavage site with multiple basic amino acids (RRRKR). This wild-type sequence makes HA more susceptible to a variety of ubiquitous proteases that may be present in the host or in the system expressing these HA. In one embodiment, removal of these amino acids may reduce the sensitivity of HA to multiple proteases. In another embodiment, the cleavage site may be mutated to remove the cleavage site (e.g., mutated to a RESR).

The invention also uses nucleic acids and polypeptides encoding NA, HA and M1. In one embodiment, the influenza NA nucleic acid or protein is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO1, 11, 31, 32, 39, 38, 46, 47, 54 or 55. In another embodiment, the influenza HA nucleic acid or protein is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO2, 10, 56, 57, 58, 27, 28, 29, 30, 37, 36, 33, 34, 35, 42, 43, 44, 45, 50, 51, 52 or 53. In another embodiment, the influenza M1 nucleic acid or protein is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO12, 40, 41, 48 or 49.

In some embodiments, the mutation comprises a change in: these changes produce silent substitutions, additions or deletions without altering the nature or activity of the encoded proteins or the manner in which they are produced. Nucleotide variants may be produced for a variety of reasons, for example to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by insect cells such as Sf9 cells). See U.S. patent publication 2005/0118191, which is incorporated herein by reference in its entirety for all purposes. Examples of optimized codon sequences of the invention are disclosed below (e.g., SEQ ID42, 44, 46, 48, 50, 52, and 54).

In addition, nucleotides can be sequenced to ensure that the correct coding region is cloned and does not contain any undesired mutations. These nucleotides can be subcloned into expression vectors (e.g., baculovirus) for expression in any cell. The above is just one example of how to clone influenza virus proteins. Those skilled in the art will appreciate that other methods are available and possible.

The invention also provides constructs and/or vectors comprising avian, pandemic and/or seasonal nucleotides encoding influenza virus structural genes including NA, M1 and/or HA. The vector may be, for example, a phage, plasmid, viral or retroviral vector. The constructs and/or vectors encoding the structural genes of avian influenza virus, pandemic influenza virus and/or seasonal influenza virus, including NA, M1 and/or HA, should be operably linked to suitable promoters, such as, by way of non-limiting example, the AcMNPV polyhedrin promoter (or other baculovirus), the lambda phage PL promoter, the e.coli (e.coli) lac, phoA and tac promoters, the SV40 early and late promoters, and the promoters of the retroviral LTRs. Other suitable promoters will be known to those skilled in the art depending on the desired host cell and/or expression rate. The expression construct will further contain transcription initiation, termination sites, and ribosome binding sites in the transcribed region for translation. The coding portion of the transcript expressed by the construct preferably comprises a translation initiation codon at the beginning of the polypeptide to be translated and a stop codon at an appropriate position at the end of the polypeptide.

The expression vector preferably comprises at least one selection marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, as well as tetracycline, kanamycin or ampicillin resistance genes for culturing E.coli and other bacteria. Preferred vectors include viral vectors such as baculoviruses, poxviruses (e.g., vaccinia virus, avipox virus (avipox virus), canarypox virus, fowlpox virus, raccoon poxvirus, swinepox virus, etc.), adenoviruses (e.g., canine adenovirus), herpesviruses, and retroviruses. Other vectors that can be used in the present invention include vectors for bacteria, including pQE70, pQE60 and pQE-9, pBluescript vector, Phagescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT 5. Preferred eukaryotic vectors include pFastBac1pWINEO, pSV2CAT, pOG44, pXT1 and pSG, pSVK3, pBPV, pMSG and pSVL. Other suitable vectors will be apparent to those skilled in the art. In one embodiment, the vector comprising nucleotides encoding structural genes of avian influenza virus, pandemic influenza virus and/or seasonal influenza virus (including HA, M1 and/or NA) is pFastBac. In another embodiment, the vector comprising an insert consisting of nucleotides encoding structural genes of avian influenza virus, pandemic influenza virus and/or seasonal influenza virus (including HA, M1 and/or NA) is pFastBac.

The recombinant vector may then be transfected, infected or transformed into a suitable host cell. Thus, the present invention provides a host cell that: they comprise a vector (or vectors) containing a nucleic acid encoding HA, M1 and/or NA and allowing expression of HA, M1 and/or NA in the host cell under conditions which allow VLPs to be formed.

In one embodiment, the above recombinant constructs may be used for transfection, infection or transformation and may express HA, NA and M1 influenza proteins in eukaryotic and/or prokaryotic cells. Eukaryotic host cells include yeast, insect, avian, plant, caenorhabditis elegans (c.elegans) (or nematodes), and mammalian host cells. Non-limiting examples of insect cells are: spodoptera frugiperda (Sf) cells, such as Sf9, Sf21, Trichoplusia ni (Trichoplusia ni) cells, such as High Five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells are Saccharomyces cerevisiae (S.cerevisiae), Kluyveromyces lactis (Kluyveromyces lactis; K.lactis), Candida species including Candida albicans (C.albicans) and Candida glabrata (C.glabrata), Aspergillus nidulans (Aspergillus nidulans), Schizosaccharomyces pombe (Schizosaccharomyces pombe; S.pombe), Pichia pastoris (Pichia pastoris), and yarrowia lipolytica (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 (HEK) cells and African green monkey cells, CV1 cells, HeLa cells, MDCK cells, Vero and Hep-2 cells. Xenopus laevis (Xenopuslaevis) oocytes, or other cells of amphibian origin, may also be used. Prokaryotic host cells include bacterial cells such as E.coli, B.subtilis, and Mycobacteria.

Vectors, such as vectors comprising HA, NA, and/or M1 polynucleotides, may be transfected into host cells according to techniques well known in the art. For example, nucleic acids can be introduced into eukaryotic cells by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagents. In one embodiment, the vector is a recombinant baculovirus. In another embodiment, the recombinant baculovirus is transfected into a eukaryotic cell. In a preferred embodiment, the cell is an insect cell. In another embodiment, the insect cell is an Sf9 cell.

In another embodiment, the vector and/or host cell comprises the nucleotides: the nucleotide encodes an avian influenza virus, pandemic influenza virus and/or seasonal influenza virus HA protein selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In another embodiment, the vector and/or host cell comprises the nucleotides: the nucleotide encodes a NA protein selected from N1, N2, N3, N4, N5, N6, N7, N8, and N9. In another embodiment, the vector and/or host cell comprises influenza HA, M1 and/or NA. In another embodiment, the vector and/or host cell consists essentially of HA, M1, and/or NA. In yet another embodiment, the vector and/or host cell consists of influenza proteins including HA, M1 and NA. These vectors and/or host cells contain HA, NA, and M1, and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (other than fragments of M1, HA, and/or NA). In another embodiment, the nucleotide encodes HA and/or NA: when they are expressed on the surface of the VLP, they exhibit hemagglutinin activity and/or neuraminidase activity, respectively.

The invention also provides constructs and methods that will increase the efficiency of VLP production. For example, cleavage sites in the protein are removed to increase protein expression (see above). Other methods include the addition of leader sequences to the HA, NA and/or M1 proteins to increase transport efficiency. For example, heterologous signal sequences can be fused to HA, NA, and/or M1 influenza proteins. In one embodiment, the signal sequence may be derived from a gene of an insect cell and fused to an influenza HA protein (for expression in an insect cell). In another embodiment, the signal peptide is a chitinase signal sequence that may work efficiently in a baculovirus expression system. In other embodiments, interchanging leader sequences between influenza proteins may provide better protein transport. For example, H5 hemagglutinin has proven to be less efficient for transport to the particle surface. Whereas H9 hemagglutinin is more efficient in targeting and integrating into the surface. Thus, in one embodiment, the H9 leader sequence is fused to the H5 protein.

Another approach to increase the efficiency of VLP production is to codon optimize the nucleotides encoding HA, NA and/or M1 proteins for a particular cell type. For example, nucleic acids are codon optimized for expression in Sf9 cells (see U.S. patent publication 2005/0118191, which is incorporated herein by reference in its entirety for all purposes). Examples of codon optimized sequences for Sf9 cells are disclosed below (e.g., SEQ ID42, 44, 46, 48, 50, 52, and 54). In one embodiment, the nucleic acid sequence of the codon-optimized influenza protein is at least 85%, 90%, 95%, 96, 97, 98 or 99% identical to any of SEQ ID NOs 42, 44, 46, 48, 50, 52 and 54.

The invention also provides a method of producing a VLP, the method comprising expressing an avian, pandemic and/or seasonal influenza protein under conditions such that a VLP can be formed. Depending on the expression system and host cell chosen, host cells transformed with the expression vector are cultured under conditions to express the recombinant protein and form VLPs, to produce the VLPs. The selection of suitable culture conditions is within the skill of one of ordinary skill in the art.

Methods of culturing cells engineered to produce the VLPs of the invention include, but are not limited to, batch, fed-batch, continuous and perfusion (perfusion) cell culture techniques. Cell culture means growing and propagating cells in a bioreactor (fermentation chamber) where the cells are propagated and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is performed in a bioreactor under conditions of sterility, controlled temperature and atmosphere. A bioreactor is a chamber (chamber) for culturing cells in which environmental conditions such as temperature, atmosphere, agitation and/or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., a bag made of plastic or plastic material)Wave Biotech, Bridgewater, NJ). In another embodiment, the pre-sterilized plastic bag is a bag of about 50L to 1000L.

These VLPs are then isolated by methods that preserve their integrity, such as by gradient centrifugation, e.g., cesium chloride, sucrose and iodixanol (iodixanol) gradient centrifugation, and standard purification techniques, including, e.g., ion exchange and gel filtration chromatography.

The following is an example of how to prepare, isolate and purify the VLPs of the invention. Typically, VLPs are produced by recombinant cell lines, wherein these cell lines are engineered to produce VLPs when grown in cell culture (see above). The production of VLPs can be achieved using the scheme shown in fig. 26. It will be appreciated by those skilled in the art that other methods may be used to prepare and purify the VLPs of the invention and the invention is therefore not limited to the methods described.

Production of VLPs of the invention Sf9 cells (uninfected) may be first inoculated into shake flasks, allowing the cells to expand and scale up as they grow (e.g. from 125ml flasks to 50L Wave bags). The medium used to culture the cells is formulated for a suitable cell line (preferably serum-free medium, e.g., insect medium ExCell-420, JRH). The cells are then infected with the recombinant baculovirus at the most efficient multiplicity of infection (e.g., about 1to about 3 plaque forming units per cell). Once infection occurs, the influenza HA, NA and M1 proteins are expressed from the viral genome, self-assemble into VLPs, and secreted from the cells approximately 24 to 72 hours after infection. Typically, when the cells are in mid-log phase of growth (4-8X 10)⁶Individual cells/ml) and at least about 90% survives, infection efficiency is highest.

The VLPs of the invention may be harvested approximately 48 to 96 hours after infection, before VLP levels in the cell culture medium approach a maximum value but extensive cell lysis has not occurred. The Sf9 cell density and viability at harvest may be: about 0.5X 10⁶From about 1.5X 10 cells/ml to⁶Individual cells/ml, viability was at least 20%, as indicated by the dye exclusion assay. The medium was then removed and clarified. NaCl may be added to the medium to a concentration of about 0.4 to about 1.0M, preferably to about 0.5M, to avoid aggregation of the VLPs. Tangential Flow Filtration (TFF) can be performed using disposable pre-sterilized hollow fiber 0.5 or 1.00 μm cartridge filters or similar devices to remove cells and cell debris from the cell culture medium containing the VLPs of the present invention.

The VLPs in the clarified culture medium can then be concentrated by ultrafiltration using a disposable, pre-sterilized 500,000 molecular weight cut-off hollow fiber cartridge filter. The concentrated VLPs may be diafiltered against 10 volumes of Phosphate Buffered Saline (PBS) ph7.0 to 8.0 containing 0.5M NaCl to remove residual media components.

The concentrated, diafiltered VLPs may be further purified by centrifugation at 6,500 Xg for 18 hours at about 4 ℃ to about 10 ℃ on a 20% -60% discontinuous sucrose gradient in 0.5M NaCl in pH7.2PBS buffer. Typically, VLPs will form a prominent visible band between about 30% to about 40% sucrose or at the interface (in step gradients of 20% and 60%), which can be collected from the gradient and stored. The product can be diluted to contain 200mM NaCl in preparation for the next step in the purification process. This product contains VLPs and may contain intact baculovirus particles.

VLPs can be further purified by anion exchange chromatography or 44% isopycnic sucrose cushion (cushion) centrifugation. In anion exchange chromatography, samples from a sucrose gradient (see above) are loaded onto a column packed with an anionic medium (e.g., Matrix Fractogel EMD TMAE) and eluted by a salt gradient (from about 0.2M to about 1.0M NaCl) that separates VLPs from other impurities (e.g., baculovirus and DNA/RNA). In the sucrose cushion method, a sample containing VLPs is added to a 44% sucrose cushion and centrifuged at 30,000g for about 18 hours. VLPs form bands at the top of 44% sucrose, while baculovirus precipitates at the bottom, other impurity proteins stay in the top 0% sucrose layer. The VLP peak or VLP band was collected.

If desired, the whole baculovirus can be inactivated. Inactivation may be by chemical means, such as formalin or beta-propiolactone (BPL). Removal and/or inactivation of whole baculovirus can also be achieved using mainly selective precipitation and chromatographic methods known in the art, as exemplified above. The inactivation method comprises incubating a sample containing VLPs in 0.2% BPL at about 25 ℃ to about 27 ℃ for 3 hours. Baculovirus can also be inactivated by incubating samples containing VLPs at 0.05% BPL for 3 days at 4 ℃ followed by 1 hour at 37 ℃.

After the inactivation/removal step, the product containing the VLPs may be subjected to another diafiltration step to remove any reagents from the inactivation step and/or any residual sucrose, and the VLPs placed in a desired buffer (e.g., PBS). The solution containing the VLPs may be sterilized by methods known in the art (e.g., sterile filtration) and stored in a refrigerator or cryo-refrigerator.

The above techniques may be implemented on a variety of scales. Such as T-flasks (T-flashes), shake flasks, roller bottles, or even industrial-scale capacity bioreactors. The bioreactor may comprise a stainless steel tank or a pre-sterilized plastic bag (e.g., the system sold by Wave Biotech, Bridgewater, NJ). The person skilled in the art will know the best choice for his purpose.

Amplification and production of baculovirus expression vectors, and infection of cells with recombinant baculovirus to produce recombinant influenza VLPs, can be achieved in insect cells, such as the Sf9 insect cells described previously. In a preferred embodiment, the cell is an SF9 cell infected with an engineered recombinant baculovirus producing an influenza VLP.

Pharmaceutical or vaccine formulations and administration

Pharmaceutical compositions useful herein comprise a VLP of the invention and a pharmaceutically acceptable carrier, wherein the carrier, including any suitable diluent or excipient, includes any agent that does not itself induce an immune response harmful to the vertebrate receiving the composition, and which can be administered without causing any abnormal toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or recorded in the U.S. pharmacopeia, european pharmacopeia, or other generally recognized pharmacopeia for use in vertebrates, and more particularly in humans. These compositions can be used as vaccines and/or antigenic compositions for inducing a protective immune response in a vertebrate.

The pharmaceutical formulation of the invention comprises VLPs comprising influenza M1, HA and/or NA proteins and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic buffered aqueous solution, and combinations thereof. Remington's pharmaceutical sciences (Mack pub. co. n. j. current edition) provides a thorough discussion of pharmaceutically acceptable carriers, diluents and other excipients. The formulation must be suitable for the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans, preferably the formulation is sterile, non-particulate and/or non-pyrogenic.

The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition may be in solid form, such as a lyophilized powder suitable for reconstitution; a liquid solution; suspending the solution; an emulsion; a tablet; pills; a capsule; a sustained release formulation; or a powder. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.

The invention also provides a pharmaceutical pack (pharmaceutical pack) or kit (kit); comprising one or more containers filled with one or more components of the vaccine formulation of the present invention. In a preferred embodiment, the kit comprises two containers, one of which contains the VLP and the other of which contains an adjuvant. The container may also be accompanied by an announcement, in the form of a regulation by a governmental agency regulating the production, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of production, use or sale for human administration.

The invention also provides for packaging of the VLP formulation in a sealed container, such as an ampoule or sachet (sachette), indicating the quantity of composition. In one embodiment, the VLP composition is provided as a liquid, in another embodiment, as a dry sterile lyophilized powder or a water-free concentrate in a sealed container, and may be reconstituted, e.g., with water or saline, to a suitable concentration for administration to a subject. Preferably, the VLP composition is provided as a dry sterile lyophilized powder in a sealed container, with a unit dose of preferably about 1 μ g, about 5 μ g, about 10 μ g, about 20 μ g, about 25 μ g, about 30 μ g, about 50 μ g, about 100 μ g, about 125 μ g, about 150 μ g or about 200 μ g. Alternatively, the unit dose of the VLP composition is less than about 1 μ g (e.g., about 0.08 μ g, about 0.04 μ g; about 0.2 μ g, about 0.4 μ g, about 0.8 μ g, about 0.5 μ g or less, about 0.25 μ g or less, or about 0.1 μ g or less), or more than about 125 μ g (e.g., about 150 μ g or more, about 250 μ g or more, or about 500 μ g or more). These doses can be measured in μ g of total VLPs or HA. The VLP composition should be administered within about 12 hours, preferably within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after reconstitution from the lyophilized powder.

In an alternative embodiment, the VLP composition is provided as a liquid in a sealed container indicating the amount and concentration of the VLP composition. Preferably, the VLP composition is provided in a sealed container at a concentration of at least about 50 μ g/ml, preferably at least about 100 μ g/ml, at least about 200 μ g/ml, at least 500 μ g/ml, or at least 1 mg/ml.

Typically, the influenza VLPs of the invention are administered in an effective amount or quantity (as defined above) sufficient to stimulate an immune response against one or more influenza virus strains. Preferably, administration of the VLP of the invention elicits significant immunity against at least one influenza virus. Typically, the dosage may be adjusted within this range depending on, for example, age, physical health, body weight, sex, diet, time of administration, and other clinical factors. Prophylactic vaccine formulations are administered systemically, for example, by subcutaneous or intramuscular injection using a needle and syringe or a needle-less injection device. Alternatively, the vaccine formulation is administered intranasally, by drops, large particle aerosol (greater than about 10 microns), or by spraying into the upper respiratory tract. Although any of the above routes of delivery produce an immune response, intranasal administration also brings the additional benefit of eliciting mucosal immunity at the site of influenza virus entry.

Thus, the invention also includes a method of formulating a vaccine or antigenic composition that induces significant immunity in a subject against influenza virus infection or at least one symptom thereof, comprising adding to the formulation an effective dose of influenza VLPs.

While it is preferred to stimulate significant immunity in a single dose, additional doses may be administered by the same or different routes to achieve the desired effect. For example, in newborns and infants, multiple administrations may be required to elicit sufficient levels of immunity. Administration may be continued at intervals throughout childhood as needed to maintain adequate levels of protection against influenza infection. Similarly, adults who are particularly susceptible to recurrent or severe influenza infections, such as health care workers, day care workers, children's families, the elderly, and individuals with impaired cardiopulmonary function, may require multiple immunizations to establish and/or maintain a protective immune response. The level of induced immunity can be monitored, for example, by measuring the amount of neutralizing secretory and serum antibodies, and adjusting the dosage or repeating the vaccination as needed to elicit and maintain the desired level of protection.

Thus, in one embodiment, a method of inducing significant immunity to an influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein the VLPs comprise influenza HA, NA, and M1 proteins. In another embodiment, a method of inducing significant immunity to an influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs consist essentially of influenza HA, NA, and M1. The VLPs may comprise negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, a method of inducing significant immunity to an influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein the VLPs consist of influenza HA, NA, and M1. In another embodiment, the influenza HA, NA and M1 are derived from seasonal influenza and/or avian influenza viruses. In another embodiment, a method of inducing significant immunity to an influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of an influenza VLP comprising an influenza protein, wherein the influenza protein consists of HA, NA, and M1 proteins. These VLPs comprise HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, the HA and/or NA exhibit hemagglutinin activity and/or neuraminidase activity, respectively. In another embodiment, the subject is a mammal. In another embodiment, the mammal is a human. In another embodiment, the method comprises inducing significant immunity against influenza virus infection or at least one symptom thereof by administering the formulation in one dose. In another embodiment, the method comprises inducing significant immunity against influenza virus infection or at least one symptom thereof by administering the formulation in multiple doses.

Methods of administration of compositions (vaccines and/or antigenic preparations) comprising VLPs 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 particular embodiment, the composition of the invention is administered intramuscularly, intravenously, subcutaneously, transdermally or intradermally. 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 mucosa, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, bladder, and intestinal mucosa, etc.), and may be administered with other biologically active agents. In some embodiments, the antibody or other immune response induced by the intranasal or other mucosal route of administration of the VLP-containing compositions of the invention may be significantly higher than other routes of administration. In another embodiment, intranasal or other mucosal routes of administration of the VLP-containing compositions of the invention may elicit antibodies or other immune responses that will elicit cross-protection against other influenza virus strains. Administration may be systemic or local.

In yet another embodiment, the vaccine and/or antigenic preparation is administered in such a way that it targets mucosal tissues to elicit an immune response at the site of immunization. For example, immunization can be targeted to mucosal tissues, such as gut-associated lymphoid tissue (GALT), by oral administration of compositions containing adjuvants with specific mucosal targeting properties. Other mucosal tissues may also be targeted, such as nasopharyngeal lymphoid tissue (NALT) and Bronchial Associated Lymphoid Tissue (BALT).

The vaccine and/or antigenic preparation of the invention may also be administered according to a dosage schedule (dosage schedule), for example a single priming administration followed by multiple boosting administrations with the vaccine composition. In particular embodiments, the second dose of the composition is administered about two weeks to one year, preferably about 1 month, about 2 months, about 3 months, about 4 months, about 5 months to about 6 months after the initial administration. Further, the third dose may be administered after the second dose, and after the initial administration for about 3 months to about two years or even longer, preferably about 4 months, about 5 months, or about 6 months, or about 7 months to about 1 year. When no or low levels of specific immunoglobulin are detected in the serum and/or urine or mucosal secretions of the subject after administration of the second dose, a third dose may optionally be administered. In a preferred embodiment, the second dose is administered about 1 month after the first administration, and the third dose is administered about 6 months after the first administration. In another embodiment, the second dose is administered about 6 months after the first administration.

In another embodiment, the VLPs of the invention may be administered as part of a combination therapy. For example, the VLPs of the invention may be co-formulated with other immunogenic compositions and/or antiviral agents, such as Amantadine (Amantadine), Rimantadine (Rimantadine), Zanamivir (Zanamivir), and oseltamivir (ostetavir).

The person skilled in the art can easily determine the dosage of the pharmaceutical preparation, e.g. by first identifying a dose effective to elicit a prophylactic or therapeutic immune response, e.g. by measuring the serum titer of virus-specific immunoglobulins or by measuring the inhibition ratio of antibodies in a serum sample or a urine sample or mucosal secretions. The dose can be determined from animal studies. A non-limiting list of animals used to study influenza viruses includes guinea pigs, Syrian hamster (Syrian hamster), chinchilla (chinchinchialla), hedgehog, chickens, rats, mice and ferrets (ferret). Most animals are not the natural host for influenza virus, but are still available for study of various aspects of the disease. For example, a vaccine candidate, e.g., a VLP of the invention, may be quantitatively administered to any of the above animals to partially characterize the induced immune response and/or to determine whether any neutralizing antibodies are produced. For example, many studies have been conducted in mouse models because mice are small in size and their low cost allows researchers to conduct large-scale studies. However, the small size of the mouse also increases the difficulty of quickly observing the clinical signs of any disease, and the mouse is not a predictive model of disease in humans.

Ferrets have been widely used for the study of human influenza virus infection and various aspects of its course of action. Without the use of ferrets, the development of many modern concepts of influenza virus immunity is not possible (Maher et al 2004). Ferrets have proven to be a good model for studying influenza for several reasons: influenza infection in ferrets closely approximates influenza infection in humans in terms of clinical signs, pathogenesis and immunity; human influenza a and b viruses naturally infect ferrets, giving people the opportunity to study a fully controlled population to observe the spread of infection, disease, and interplay between amino acid sequence variations in the influenza glycoprotein; and ferrets also have other constitutional features that make them ideal models for the interpretation of the disease manifestations. For example, ferrets and humans display very similar clinical signs of influenza infection, which appear to depend on host age, viral strain, environmental conditions, the extent of secondary bacterial infection, and many other variables. Thus, the efficacy of an influenza vaccine from the ferret model can be more readily correlated to a dosage regimen by one skilled in the art than to a mouse or any of the other models described above.

In addition, one skilled in the art can conduct human clinical studies to determine the preferred effective dose for humans. Such clinical studies are routine and well known in the art. The exact dosage to be used will also depend on the route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal assay systems.

It is also well known in the art that non-specific stimulators of the immune response, referred to as adjuvants, can be used to enhance the immunogenicity of a particular composition. Adjuvants have been used experimentally to promote a general increase in immunity to unknown antigens (see U.S. patent No.4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and thus adjuvants are well known to those of ordinary skill in the art. Some adjuvants affect the manner of antigen presentation. For example, when protein antigens are precipitated by alum, the immune response is increased. Emulsification of the antigen can also extend the duration of antigen presentation. The scope of the present invention is intended to cover the incorporation of any adjuvant described in Vogel et al "a complex of Vaccine Adjuvants and Excipients (2nd Edition)", the entire contents of which are incorporated herein by reference for all purposes.

Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). It is also envisaged to use RIBI, which contains 3 ingredients extracted from the bacteria in a 2% squalene/Tween 80 emulsion: MPL, Trehalose Dimycolate (TDM), and Cell Wall Skeleton (CWS). MF-59, may also be used,An MHC antigen.

In one embodiment of the invention, the adjuvant is a low lamellar (paucilamellar) lipid vesicle having about 2 to 10 bilayers arranged as a plurality of substantially spherical shells separated by an aqueous layer surrounding a lipid bilayer free lipid bilayerThe central large cavity is shaped. The few-lamellar lipid vesicles can act to stimulate an immune response in several ways: as non-specific stimulators, as antigen carriers, as carriers for other adjuvants, and combinations thereof. For example, when a vaccine is prepared by mixing an antigen with pre-formed vesicles such that the antigen remains extracellular (extracellular) with respect to the vesicles, the few lamellar lipid vesicles act as non-specific immune stimulators. By encapsulating the antigen in the central cavity of the vesicle, the vesicle serves both as an immunostimulant and as an antigen carrier. In another embodiment, the vesicles consist essentially of non-phospholipid vesicles. In another embodiment, the vesicle is Novasomes.Is a few lamellar non-phospholipid vesicles ranging from about 100nm to about 500 nm. They comprise Brij72, cholesterol, oleic acid, and squalene. Novasomes have been shown to be effective adjuvants for influenza antigens (see us patents 5,629,021, 6,387,373 and 4,911,928, the entire contents of which are incorporated herein by reference for all purposes).

In one aspect, agents are used to achieve the adjuvant effect, such as alum, which is used as a solution of about 0.05 to about 0.1% in phosphate buffered saline. Alternatively, VLPs can be made with synthetic carbohydrate polymersThe polymer is used as an approximately 0.25% solution. Some adjuvants, such as certain organic molecules obtained from bacteria, act on the host rather than the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [ MDP ]]) A bacterial peptidoglycan. In other embodiments, hemocyanin and hemoglobin (hemoerythrin) may also be used with the VLPs of the invention. In certain embodiments it is preferred to use a key hole from the keywayHemocyanin (KLH), but others may be usedMollusc and arthropod hemocyanin and hemoglobin.

Various polysaccharide adjuvants may also be used. For example, the utility of various pneumococcal polysaccharide adjuvants on mouse antibody responses has been described (Yin et al, 1989). The dose that produces the best response or that otherwise does not produce inhibition should be used as indicated (Yin et al, 1989). Polyamine versions of polysaccharides are particularly preferred, such as chitin and chitosan, including chitosan. In another embodiment, lipophilic disaccharide-tripeptide derivatives of muramyl dipeptides are described for use in artificial liposomes formed from phosphatidylcholine and phosphatidylglycerol.

Amphiphilic substances and surface active substances, such as saponins and derivatives such as QS21(cambridge biotech) constitute another class of adjuvants for VLPs of the invention. Nonionic block copolymer surfactants (Rabinovich et al, 1994) may also be used. Oligonucleotides are another useful class of adjuvants (Yamamoto et al, 1988). Quil A and lentinan (lentinen) are other adjuvants that may be used in certain embodiments of the present invention.

Another class of adjuvants are detoxified endotoxins, such as the refined detoxified endotoxins of U.S. patent No.4,866,034. These purified detoxified endotoxins are effective in producing adjuvant responses in vertebrates. Of course, detoxified endotoxin can be used in combination with other adjuvants to make multi-adjuvant formulations. For example, combinations of detoxified endotoxin with trehalose dimycolate are specifically contemplated, as described in U.S. patent No.4,435,386. Also contemplated are combinations of detoxified endotoxin with trehalose dimycolate and endotoxin glycolipids (U.S. Pat. No.4,505,899), as well as combinations of detoxified endotoxin with Cell Wall Scaffolds (CWS) or with CWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. We envision that only the combination of CWS and trehalose dimycolate without detoxified endotoxin would be useful as well, as described in us patent No.4,520,019.

Those skilled in the art will be aware of the different classes of adjuvants that can be conjugated to the vaccine according to the present invention, these include Alkyl Lysophospholipids (ALP); BCG; and biotin (including biotinylated derivatives), among others. Some adjuvants particularly contemplated for use herein are teichoic acids from Gram-cells (Gram-cells). These include lipoteichoic acids (LTA), Ribitol Teichoic Acids (RTA) and Glycerol Teichoic Acids (GTA). Active forms of their synthetic counterparts may also be used in the present invention (Takada et al, 1995).

For example, if it is desired to elicit antibodies or subsequently obtain activated T cells, various adjuvants, even those not normally used in humans, may still be used in other vertebrates. Toxicity or other adverse effects which may be caused by adjuvants or cells, such as may occur with unirradiated tumor cells, are irrelevant in this case.

Another method of inducing an immune response may be achieved by formulating the VLPs of the invention with an "immunostimulant". These "immune stimulators" are the body's own chemical messengers (cytokines) that increase the immune system response. Immunostimulatory substances include, but are not limited to, various cytokines, lymphokines, and chemokines, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13), having immunostimulatory, immunopotentiating, and proinflammatory activities; 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, and the like. The immunostimulatory molecule may be administered in the same formulation as the influenza VLP, or may be administered separately. The protein or expression vector encoding the protein may be administered to produce an immunostimulatory effect.

Methods of stimulating an anti-influenza immune response

The VLPs of the invention may be used to prepare compositions that stimulate an immune response that confers immunity or significant immunity against influenza virus. Both mucosal and cellular immunity can contribute to immunity against influenza infection and disease. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infections. Secretory immunoglobulin a (siga) is involved in the protection of the upper respiratory tract and serum IgG in the protection of the lower respiratory tract. The immune response elicited by the infection is resistant to reinfection with the same virus or antigenically similar strains of virus. Influenza viruses frequently undergo unpredictable changes, and thus, following natural infection, host immunity may provide effective protection against new strains circulating in the human population for only a few years.

The VLPs of the invention may induce significant immunity in a vertebrate (e.g. a human) when administered to said vertebrate. The significant immunity results from an immune response against the influenza VLPs of the invention, protecting or ameliorating influenza virus infection or at least alleviating symptoms of influenza virus infection in said vertebrate. In some cases, if the vertebrate is infected, the infection will be asymptomatic. The response may not be a fully protective response. In this case, if the vertebrate is infected with influenza virus, the vertebrate will experience reduced symptoms or a shorter duration of symptoms compared to a non-immunized vertebrate.

In one embodiment, the invention includes a method of inducing significant immunity to an influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of influenza VLPs. In another embodiment, the induction of significant immunity reduces the duration of influenza symptoms. In another embodiment, a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs comprise influenza HA, NA, and M1 proteins. In another embodiment, the influenza VLP comprises an influenza protein, wherein the influenza protein consists of HA, NA, and M1 proteins. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs consist essentially of HA, NA, and M1. The VLPs may comprise negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs consist of influenza HA, NA, and M1. In another embodiment, the HA and/or NA exhibit hemagglutinin activity and/or neuraminidase activity, respectively. In another embodiment, the subject is a mammal. In another embodiment, the mammal is a human. In yet another embodiment, the VLP is formulated with an adjuvant or an immunostimulant.

Recently, efforts have been made jointly to construct vaccines against avian influenza viruses that have the potential to produce pandemics. This is because many avian influenza viruses have crossed species barriers to directly infect humans, causing the human to become sick and in some cases leading to death. These viruses are H5N1, H9N2 and H7N7(Coxet al, 2004). A recent study examined the potential of using inactivated H5N1 influenza virus as a vaccine. The formula of the vaccine is similar to the existing licensed inactivated vaccine which is licensed to be on the market. The conclusion of this study was that the use of inactivated H5N1 virus did induce an immune response in humans, but the doses given were high (90 μ g avian influenza compared to 15 μ g of licensed vaccine) (Treanor et al, 2006). A large number of avian influenza antigens are impractical for vaccination programs worldwide. As explained below, the VLP of the invention elicits an immune response in a vertebrate when administered to said vertebrate.

Accordingly, the present invention encompasses a method of inducing significant immunity against an influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of avian influenza VLPs. In another embodiment, the induction of significant immunity reduces the duration of influenza symptoms. In another embodiment, the induction of immunity is from administration of a VLP of the invention comprising at least 0.2 μ g avian HA. In another embodiment, the induction of immunity is from administration of the VLP of the invention comprising at least 0.2 μ g avian HA to at least 15 μ g avian HA. Administration may be divided into one or more doses, but advantageously is carried out in one dose. In another embodiment, the VLP avian HA is derived from avian influenza H5N 1.

In another embodiment, the invention includes a method of inducing significant immunity to an avian influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of an avian influenza VLP, wherein said VLP comprises avian influenza HA, NA and M1. In another embodiment, the avian influenza VLP comprises an avian influenza protein, wherein the avian influenza protein consists of HA, NA, and M1 proteins. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, the method of inducing significant immunity to an avian influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of an avian influenza VLP, wherein said VLP consists essentially of avian influenza HA, NA and M1. The VLPs may comprise negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, the method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs consist of avian influenza HA, NA and M1 proteins. In another embodiment, the avian influenza HA and NA are each H5N 1. In another embodiment, the avian influenza HA and NA are each H9N 2. In another embodiment, the avian influenza HA and NA are each H7N 7. In another embodiment, the avian influenza HA and/or NA exhibits hemagglutinin activity and/or neuraminidase activity, respectively. In another embodiment, the subject is a mammal. In another embodiment, the mammal is a human. In yet another embodiment, the VLP is formulated with an adjuvant or an immunostimulant.

In another embodiment, the avian influenza VLP induces an immune response in a vertebrate that is about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 128-fold (or higher) more potent than a similar avian influenza antigen formulated in a manner similar to existing licensed inactivated vaccines. Existing formulations include whole inactivated virus (e.g. formaldehyde treated) vaccines, split (split) virus (chemically disrupted) vaccines, and subunit (purified glycoprotein) vaccines. Methods for determining the efficacy of vaccines are known and routine in the art. For example, a microneutralization assay and a coagulation inhibition assay can be performed to determine the efficacy of an avian VLP vaccine relative to an avian influenza antigen formulated in a manner similar to existing licensed inactivated vaccines. In one embodiment, the above increase in potency is achieved when administering to a vertebrate about 0.2 μ g, about 0.4 μ g, about 0.6 μ g, about 0.8 μ g, about 1 μ g, about 2 μ g, about 3 μ g, about 4 μ g, about 5 μ g, about 6 μ g, about 7 μ g, about 9 μ g, about 10 μ g, about 15 μ g, about 20 μ g, about 25 μ g, about 30 μ g, about 35 μ g, 40 μ g, about 45 μ g, about 50 μ g or more VLPs and formulated in a manner similar to the antigens of existing licensed commercial VLP inactivated vaccines (i.e., a middle amount of HA and/or NA and an equivalent amount of HA and/or NA formulated in a manner similar to that of a licensed inactivated vaccine and/or any other antigen). Can be measured in terms of HA content. For example, 1 μ g of the VLPs of the invention is 1 μ g of HA in a solution of the VLPs containing HA or can be measured by VLP weight.

People receive seasonal influenza vaccines every year to reduce the incidence of annual influenza cases. Currently, there are two subtypes of influenza a and influenza b epidemics in the united states. Thus, existing vaccines are trivalent to provide protection against circulating viral strains. Different influenza virus strains or influenza virus variants change each year. Thus, new vaccine compositions are manufactured and administered for the majority of years. Inactivated vaccines are produced by amplifying the virus in embryonated chicken eggs. Allantoic fluid is collected, concentrated and virus purified for inactivation. Thus, existing licensed influenza virus vaccines may contain trace amounts of residual egg proteins and therefore should not be administered to people with allergic hypersensitivity to eggs. Furthermore, the supply of eggs must be organized and the virus strain used for vaccine production must be selected months before the next flu season, limiting the flexibility of the process, often resulting in delays and shortages in production and distribution. Furthermore, some influenza strains replicate poorly in embryonated chicken eggs, which may limit the influenza strains that can be grown and formulated into vaccines.

As mentioned above, the production of VLPs of the invention does not require eggs. These VLPs are prepared by cell culture systems. Accordingly, the present invention encompasses a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of seasonal influenza VLPs. As discussed above, seasonal influenza virus refers to an influenza virus strain that has been determined to be spreading within a population for a certain influenza season based on epidemiological investigations conducted by national influenza centers throughout the world. The results of the study, as well as some isolated influenza viruses, were sent to one of four reference laboratories of the World Health Organization (WHO) for detailed study, one of which was located at the disease control and prevention center (CDC) of atlanta. These laboratories tested how well antibodies prepared against existing vaccines reacted with circulating viruses and new influenza viruses. This information and information about flu activity were aggregated and submitted to an advisory board of the U.S. Food and Drug Administration (FDA) and at the WHO's conference. The result of these conferences was the selection of three viruses (two subtypes of influenza a virus and one subtype of influenza b virus) into an influenza vaccine for the next fall and winter. The selection was made in the northern hemisphere in month 2 and in the southern hemisphere in month 9. Typically, one or two of the three strains in a vaccine are changed annually. In another embodiment, the induction of significant immunity reduces the duration of influenza symptoms.

In another embodiment, the invention comprises a method of inducing significant immunity to a seasonal influenza virus infection or at least one symptom thereof in a subject, comprising administering at least one effective dose of a seasonal influenza VLP, wherein the VLP comprises seasonal influenza HA, NA, and M1 proteins. In another embodiment, the seasonal influenza VLP comprises a seasonal influenza protein, wherein the influenza protein is comprised of HA, NA, and M1 proteins. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, the method of inducing significant immunity to a seasonal influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of a seasonal influenza VLP, wherein the VLP consists essentially of seasonal influenza HA, NA, and M1. The VLPs may comprise negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs consist of seasonal influenza HA, NA, and M1. In another embodiment, the avian influenza HA and/or NA exhibits hemagglutinin activity and/or neuraminidase activity, respectively. In another embodiment, the subject is a mammal. In another embodiment, the mammal is a human. In yet another embodiment, the VLP is formulated with an adjuvant or an immunostimulant.

Typically, the seasonal influenza VLPs of the invention are administered in an amount sufficient to stimulate significant immunity against one or more seasonal influenza virus strains. In one embodiment, the VLPs are blended with other VLPs comprising different influenza subtype proteins (as listed above). In another embodiment, the formulation is a trivalent formulation comprising a mixture of VLPs having seasonal influenza HA and/or NA proteins from at least two influenza a subtypes and/or at least one influenza b subtype. In another embodiment, the subtype b is prepared by the same method as described above. In another embodiment, the multivalent composition comprises one or more VLPs of the invention as described above.

In another embodiment, the VLPs of the invention (avian or seasonal VLPs) may elicit an immune response that will provide protection against more than one influenza virus strain. Cross-protection of vertebrates by influenza VLPs constructed from a particular strain of a particular subgroup can induce cross-protection against influenza viruses of different strains and/or subgroups. The examples below show that the VLPs of the invention are capable of inducing cross-reactivity with different virus strains and/or subclasses.

The humoral immune system produces antibodies against different influenza antigens, of which HA-specific antibodies are most important for neutralizing the virus and thus preventing diseases. NA-specific antibodies are less effective at preventing infection, but they reduce virus release from infected cells. Mucosal tissues are the major entry portal for many pathogens, including influenza, while the mucosal immune system provides the first line of defense against infection in addition to innate immunity. SIgA, and to some extent IgM, is the primary neutralizing antibody against mucosal pathogens, preventing pathogen entry, and can act intracellularly to inhibit viral replication. Nasal secretions contain neutralizing antibodies, particularly against influenza HA and NA, which are predominantly of the IgA isotype and are produced locally. During primary infection, all three major Ig classes (IgG, IgA, and IgM) specific for HA can be detected in nasal washes by elisa, although IgA and IgM are detected more often than IgG. IgA, and to some extent also IgM, are actively secreted locally, while IgG is derived as serum secretion. In subjects with a local IgA response, a serum IgA response was also observed. Local IgA responses stimulated by natural infection persist for at least 3-5 months and influenza-specific IgA-directed memory cells can be detected locally. IgA is also the predominant Ig isotype in local secretions after secondary infection, and IgA responses are detected in the serum upon subsequent infection. The presence of locally produced neutralizing antibodies induced by live virus vaccines correlates with infection resistance and disease status following wild-type virus challenge.

Resistance or disease to influenza infection is associated with local and/or serum levels of antibodies to HA and NA. Serum anti-HA antibodies are the most frequently measured indicators of relevance for protection against influenza (Cox et al, 1999). Protective serum antibody (coagulation inhibition (HI) titer ≧ 40) responses were detectable in approximately 80% of subjects following natural influenza infection. B cells producing all three major Ig classes are present in normal subjects (Cox et al, 1994) and individuals who are experiencing influenza infection. In humans, serum antibodies play a role in resistance and recovery from influenza infection. Serum antibody levels against HA and NA in humans can be correlated with disease resistance following experimental and natural infection. During primary infection, three major Ig classes are detectable within 10-14 days. IgA and IgM levels peaked after 2 weeks and then began to decrease, while IgG levels peaked at 4-6 weeks. While IgG and IgM are predominant in the primary response, IgG and IgA predominate in the secondary immune response.

Thus, the invention comprises a method of inducing a significant protective antibody response against an influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of influenza VLPs. In another embodiment, induction of the significant protective antibody response reduces the duration of influenza symptoms. In another embodiment, a method of inducing a significant protective antibody response against an influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein the VLPs comprise influenza HA, NA, and M1 proteins.

In another embodiment, the invention includes a method of inducing a significant protective antibody response against an influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of influenza VLPs, wherein said VLPs consist essentially of influenza HA, NA, and M1. The VLPs may comprise negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, the VLP comprises an influenza protein, wherein the influenza protein consists of HA, NA, and M1. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs consist of influenza HA, NA, and M1. In another embodiment, wherein the influenza HA, NA and M1 are derived from seasonal influenza and/or avian influenza. In another embodiment, the HA and/or NA exhibit hemagglutinin activity and/or neuraminidase activity, respectively. In another embodiment, the subject is a mammal. In another embodiment, the mammal is a human. In yet another embodiment, the VLP is formulated with an adjuvant or an immunostimulant.

As used herein, the term "antibody" is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or immunoglobulin gene fragments. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes and the 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 type of immunoglobulin: IgG, IgM, IgA, IgD and IgE. A typical immunoglobulin (antibody) structural unit is composed of a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" (about 50-70kD) chain. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids, which is primarily responsible for antigen recognition. Antibodies exist as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases.

Cell-mediated immunity also plays a role in the recovery from influenza infection and may also prevent influenza-related complications. Influenza-specific cellular lymphocytes have been detected in the blood and lower respiratory secretions of infected subjects. Cytolysis of influenza-infected cells is mediated by CTL in conjunction with influenza-specific antibodies and complement. In infected or vaccinated individuals, the primary cytotoxic response can be detected in the blood after 6-14 days and disappeared before day 21 (Ennis et al, 1981). Influenza-specific CTLs exhibit cross-reactivity specificity in vitro culture, so that they lyse cells infected with the same type of influenza but not other types (e.g., influenza a but not influenza b virus). CTLs recognizing the internal nonglycosylated proteins, M, NP and PB2, have been isolated (Fleischer et al, 1985). CTL responses are cross-reactive among influenza a virus strains (Gerhard et al, 2001) and are important in minimizing virus spread in conjunction with antibodies (Nguyen et al, 2001).

Cell-mediated immunity also plays a role in the recovery from influenza infection and may also prevent influenza-related complications. Influenza-specific cellular lymphocytes have been detected in the blood and lower respiratory secretions of infected subjects. Cytolysis of influenza-infected cells is mediated by CTL in conjunction with influenza-specific antibodies and complement. In infected or vaccinated individuals, the primary cytotoxic response can be detected in the blood after 6-14 days and disappeared before day 21 (Ennis et al, 1981). Influenza-specific CTLs exhibit cross-reactivity specificity in vitro culture, so that they lyse cells infected with the same type of influenza but not other types (e.g., influenza a but not influenza b virus). CTLs recognizing the internal nonglycosylated proteins, M, NP and PB2, have been isolated (Fleischer et al, 1985). CTL responses are cross-reactive among influenza a virus strains (Gerhard et al, 2001) and are important in minimizing virus spread in conjunction with antibodies (Nguyen et al, 2001). .

Thus, the invention includes a method of inducing a significant protective cellular immune response against an influenza virus infection or at least one symptom thereof in a subject comprising administering at least one effective dose of influenza VLPs. In another embodiment, a method of inducing significant immunity to influenza virus infection or at least one symptom thereof in a subject comprises administering at least one effective dose of influenza VLPs, wherein said VLPs consist of influenza HA, NA, and M1. In another embodiment, the influenza VLP comprises an influenza protein, wherein the influenza protein consists of HA, NA, and M1 proteins. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, wherein said influenza HA, NA and M1 are derived from seasonal influenza and/or avian influenza viruses. In another embodiment, the HA and/or NA exhibit hemagglutinin activity and/or neuraminidase activity, respectively. In another embodiment, the subject is a mammal. In another embodiment, the mammal is a human. In yet another embodiment, the VLP is formulated with an adjuvant or an immunostimulant.

As described above, the VLPs of the invention (e.g., avian influenza and/or seasonal influenza VLPs) prevent or reduce at least one symptom of influenza infection in a subject. Symptoms of influenza are well known in the art. They include fever, myalgia, headache, severe malaise, dry cough, sore throat, weight loss and rhinitis. Thus, the methods of the invention comprise preventing or alleviating at least one symptom associated with influenza virus infection. The reduction of symptoms can be determined subjectively or objectively, e.g., by the subject self-assessment, by a physician, or by conducting a suitable assay or measurement (e.g., body temperature), including, for example, quality of life assessment, slowing of progression of influenza infection or other symptoms, reduction in severity of influenza symptoms, or a suitable assay (e.g., antibody titer (titer) and/or T cell activation assay). Objective evaluations include both animal and human evaluations.

The main influenza control strategy advocated by the counseling Committee for Immunization administration on Immunization Practices (ACIP) is vaccination of persons at risk of serious complications of influenza, particularly persons greater than or equal to 65 years old. However, annual influenza epidemics have not been alleviated and put a significant health and financial burden on our society (Glaser et al, 1996). In the past 20 years (1976-1999), a significant increase in influenza-associated all cause excess deaths has occurred. The number of influenza-associated all-cause deaths each year is over 50,000 from 1990 to 1999 (Thompson et al, 2003). Although vaccine coverage has increased to 65% in people aged 65 or older over the past 10 years, there has been no corresponding reduction in influenza-associated all-cause excess deaths.

Thus, another strategy for influenza prevention and control is the general vaccination of healthy children and individuals. The influenza infection rate, the hospitalization disease rate due to influenza and the hospitalization rate of children are all high (Neuzil et al, 2000). Children play an important role in the spread of influenza in schools, homes and communities. Vaccination of approximately 80% of schoolchildren in a community with existing influenza vaccines reduces respiratory disease in adults and excessive mortality in the elderly (Reichert et al, 2001). This concept is called community immunity or "herd immunity" and is considered to be of great importance in protecting communities from disease. Because vaccinated people have antibodies that neutralize influenza virus, their likelihood of transmitting influenza virus to others is greatly reduced. Thus, even persons who have not been vaccinated (and who have been vaccinated weakened or whose vaccine is not fully effective) can often be protected against community immunity because the persons vaccinated around them are ill. Population immunity is more effective as the percentage of vaccinated people increases. It is believed that in order to achieve population immunity, approximately 95% of people in the community must be protected by the vaccine. Non-immunized people may increase their chances of contracting the disease with others.

Accordingly, the present invention includes a method of inducing significant protective immunity against influenza virus infection in a human population or community by administering the VLPs of the invention to the human population in the community, to reduce the incidence of influenza virus infection in immunocompromised or unvaccinated individuals. In one embodiment, most school-age children are immunized against influenza virus by administering the VLPs of the invention. In another embodiment, a majority of healthy individuals in the community are immunized against influenza virus by administering the VLP of the invention. In another embodiment, the VLP of the invention is part of a "dynamic vaccination" strategy. Dynamic vaccination refers to the robust production of low potency vaccines that are associated with newly-developed pandemic strains but may not provide complete protection in mammals due to antigenic drift (see Germann et al, 2006). Due to the uncertainty of the identity of future pandemic strains, it is almost impossible to stock well-matched pandemic strains. However, vaccination with a less compatible but potentially effective vaccine may delay the spread of pandemic viruses and/or reduce the severity of symptoms of pandemic strains of influenza virus.

The invention also encompasses a vaccine comprising influenza VLPs, wherein the vaccine, when administered to a subject, induces significant immunity against influenza virus infection or at least one symptom thereof. In another embodiment, the induction of significant immunity reduces the duration of influenza symptoms. In another embodiment, the vaccine induces significant immunity in a subject against influenza virus infection or at least one symptom thereof, comprising VLPs comprising influenza HA, NA, and M1 proteins. In another embodiment, one such vaccine induces significant immunity to influenza virus infection or at least one symptom thereof in a subject, comprising a VLP consisting essentially of influenza HA, NA, and M1 proteins. The VLPs may contain negligible concentrations of other influenza proteins and/or protein impurities. In another embodiment, one such vaccine induces significant immunity to influenza virus infection or at least one symptom thereof in a subject, comprising a VLP consisting of influenza HA, NA, and M1 proteins. In another embodiment, one such vaccine induces significant immunity to influenza virus infection or at least one symptom thereof in a subject, comprising a VLP comprising an influenza protein, wherein the influenza protein consists of HA, NA, and M1 proteins. These VLPs contain HA, NA and M1 and may contain other cellular components such as cellular proteins, baculovirus proteins, lipids, carbohydrates, etc., but no other influenza proteins (except fragments of M1, HA and/or NA). In another embodiment, the influenza HA, NA and M1 proteins are derived from avian influenza and/or seasonal influenza viruses. In another embodiment, the HA and/or NA exhibit hemagglutinin activity and/or neuraminidase activity, respectively. In another embodiment, the subject is a mammal. In another embodiment, the mammal is a human. In yet another embodiment, the VLP is formulated with an adjuvant or an immunostimulant. In another embodiment, the vaccine is administered to a mammal. In yet another embodiment, the mammal is a human.

The invention is further illustrated by the following examples, which should not be construed as limiting. All references, patents and published patent applications cited in this application, as well as the figures and sequence listing, are incorporated herein by reference.

Examples

Example 1

Materials and methods

Avian influenza A/hong Kong/1073/99 (H9N2) virus HA, NA and M1 genes were expressed in Spodoptera frugiperda cells (Sf-9S cell line; ATCCPTA-4047) using a baculovirus bacmid vector expression system. HA. The NA and M1 genes were synthesized by reverse transcription and Polymerase Chain Reaction (PCR) using RNA isolated from avian influenza a/hong kong/1073/99 (H9N2) virus (fig. 1, 2 and 3). Reverse transcription and PCR used oligonucleotide primers specific for the avian influenza a/hong kong/1073/99 (H9N2) virus HA, NA and M1 genes (table 1). First, cDNA copies of these genes were cloned into the bacterial subcloning vector pCR2.1TOPO. From the three resulting plasmids based on pcr2.1topo, the HA, NA and M1 genes were inserted downstream of the AcMNPV polyhedrin promoter in the baculovirus transfer vector pFastBac1(InVitrogen) to yield three plasmids based on pFastBac 1: pHA, pNA and pM1, which express these influenza virus genes, respectively. Then, a single plasmid pHAM based on pFastBac1 was constructed encoding both HA and M1 genes, which are located downstream of different polyhedrin promoters, respectively (FIG. 4). The nucleotide sequence of the NA gene and adjacent 5 '-and 3' -regions (SEQ ID NO:1) in the pNA plasmid was determined (FIG. 1). Meanwhile, the nucleotide sequences of the HA and M1 genes and the adjacent regions (SEQ ID NOS: 2 and 3) were also determined using the pHAM plasmid (FIGS. 2 and 3).

Finally, a restriction DNA fragment encoding the HA and M1 expression cassettes from the pHAM plasmid was cloned into the pNA plasmid. This gave plasmid pNAHAM (fig. 4) encoding the HA, NA and M1 genes of avian influenza a/hong kong/1073/99 (H9N2) virus.

The plasmid pNAHAM was used to construct recombinant baculoviruses containing the influenza NA, HA and M1 genes integrated into the genome, downstream of the different baculovirus polyhedrin promoters, respectively. The resulting recombinant baculovirus was used to infect permissive Sf-9S insect cells, and as a result, the three influenza genes were co-expressed in each Sf-9S cell infected with these recombinant baculoviruses.

Expression products in Sf-9S cells were characterized 72 hours post infection (p.i.) by SDS-PAGE analysis, coomassie blue protein staining and Western immunoblot analysis using HA-specific and M1-specific antibodies (fig. 5). Western immunoblot analysis was performed using either rabbit antibodies raised against influenza virus type a/hong kong/1073/99 (H9N2) (CDC, Atlanta, Ga., USA) or mouse monoclonal antibodies against influenza M1 protein (Serotec, UK). Western immunoblot analysis detected HA, NA and M1 proteins (64 kd, 60kd and 31kd, respectively) with the desired molecular weights. The NA protein showed lower reactivity with rabbit serum against influenza a/hong kong/1073/99 (H9N2) virus compared to the amount of HA protein detected in this assay. Explanations for the amount of detectable NA protein include: the expression level of NA protein was lower in Sf-9S cells infected with recombinant baculovirus compared to HA protein; NA is less reactive with this serum under denaturing conditions in Western immunoblot assays (due to loss of important NA epitopes during membrane-bound gel electrophoresis); lower NA-antibody affinity compared to HA-antibody; or low abundance of NA-antibodies in serum.

Influenza virus detection was also performed on media from Sf-9S cells infected with recombinant baculovirus expressing HA, NA and M1 proteins from type a/hong kong/1073/99 (H9N 2). The clarified culture supernatant was subjected to ultracentrifugation at 27,000rpm to concentrate high molecular weight protein complexes of influenza virus, such as subviral particles, VLPs, VLP complexes, and other self-assembled particulate matter consisting of influenza HA, NA, and M1 proteins, if present. The centrifugally precipitated protein product was resuspended in phosphate buffered saline (PBS, ph7.2) and then further purified by ultracentrifugation over a discrete 20-60% sucrose step gradient. Fractions were collected from the sucrose gradient and analyzed using SDS-PAGE analysis, Western immunoblot analysis, and electron microscopy.

Influenza HA and M1 proteins with the desired molecular weights were detected in multiple sucrose density gradient fractions by coomassie blue staining and Western immunoblot analysis (fig. 6, table 1). This suggests that influenza virus proteins from infected Sf-9S cells are aggregated in large molecular weight complexes (e.g., capsomeric structures, subviral particles, VLPs, and/or VLP complexes). Coomassie blue staining and Western immunoblot analysis were not stable to NA protein detection, probably due to the inability of rabbit anti-influenza sera to recognize denatured NA protein in Western immunoblot analysis, but stably detected NA protein in neuraminidase activity assays (fig. 10).

TABLE 1

Fractions from a 20-60% sucrose gradient

Negative control

Positive control

The presence of high molecular weight VLPs was confirmed by gel filtration chromatography. Aliquots from the sucrose density gradient fractions containing the influenza proteins were loaded onto a Sepharose CL-4B column and fractionated by mass. The column was calibrated with blue dextran 2000(dextran blue2000), yellow dextran (dextran yellow) and vitamin B12(Amersham Pharmacia) having apparent molecular weights of 2,000,000, 20,000 and 1,357 daltons, respectively, and the column's external water volume was determined. As expected, high molecular weight influenza virus proteins migrate in the outer water volume of the column, which is characteristic of large molecular proteins, such as virus particles. Fractions were analyzed by Western immunoblot analysis to detect influenza and baculovirus proteins. For example, M1 protein was detected in the outer water volume fraction, which also contained baculovirus protein (fig. 7).

The morphology of influenza VLPs and proteins in the sucrose gradient fractions was elucidated by electron microscopy. For negative staining electron microscopy, influenza proteins from both sucrose density gradient fractions were fixed with 2% glutaraldehyde in PBS ph 7.2. Electron microscopy was performed on the negatively stained sample and the presence of macromolecular protein complexes or VLPs was found in both fractions. These VLPs exhibit different sizes (including diameters of approximately 60 and 80nm) and morphologies (spheres). Larger complexes of these two particles were also detected as well as rod-shaped particles (fig. 8). All macromolecular structures observed had spikes (enveloped particles) on their surface, which are characteristic of influenza viruses. Since the 80nm particles are similar in size and appearance to wild-type influenza virus particles, these structures are likely VLPs with significant similarity to wild-type influenza virions, including similar particle geometry, architecture, number of trigonometric divisions, symmetry, and other features. Smaller particles of about 60nm are likely subviral particles, differing from VLPs both morphologically and structurally. Similar phenomena have been reported for recombinant macromolecular proteins of different sizes and morphologies for other viruses. For example, the hepatitis b virus recombinant core antigen (HBcAg) forms particles of different sizes, which have different structures with a T-4 and T-3 triangle division, respectively (Crowther et al, 1994).

To characterize the functional properties of purified influenza a/hong kong/1073/99 (H9N2) VLPs, samples were tested in the hemagglutination assay (fig. 9) and the neuraminidase enzyme assay (fig. 10). For hemagglutination assays, a two-fold dilution of purified influenza VLPs was mixed with 0.6% guinea pig erythrocytes and incubated at 4 ℃ for 1 hour or 16 hours. The extent of clotting was visually observed and the highest dilution of recombinant influenza protein capable of agglutinating erythrocytes was determined and recorded (fig. 9). Likewise, many fractions from the sucrose density gradient showed clotting activity, suggesting the presence of various macromolecular and monomeric forms of influenza proteins. The highest titer was measured to be 1: 4000. In a control experiment, wild-type influenza A/Shangdong virus showed a titer of 1: 2000. This clotting assay showed that recombinant VLPs composed of influenza a/hong kong/1073/99 (H9N2) virus HA, NA and M1 proteins were functionally active. This suggests that the assembly, conformation and folding of the HA subunit proteins within these VLPs is similar or identical to that of wild-type influenza virus.

In addition, neuraminidase assays were performed on samples of purified H9N2 VLPs. The amount of neuraminidase activity in the sucrose density gradient fractions was determined using fetuin as substrate. In this neuraminidase assay, neuraminidase cleaves sialic acid from a substrate molecule, thereby releasing sialic acid for assay. The enzyme activity was stopped by addition of arsenite reagent. The amount of sialic acid released was determined chemically with thiobarbituric acid, which produced a pink colour proportional to the amount of free sialic acid. The amount of color (chromophore) was measured spectrophotometrically at a wavelength of 549 nm. Using this method, neuraminidase activity was shown in the sucrose gradient fraction containing influenza VLPs (fig. 10). As expected, activity was observed in several fractions, including two peak fractions. Wild-type influenza virus was used as a positive control. Wild-type influenza viruses exhibit neuraminidase activity similar to purified influenza VLPs. These findings support HA results in terms of protein conformation, suggesting that purified VLPs of influenza a/hong kong/1073/99 (H9N2) virus are functionally similar to wild-type influenza virus.

The results of the above analysis and assay indicate that the expression of influenza a/hong kong/1073/99 (H9N2) HA, NA and M1 proteins is sufficient for the self-assembly and transport of functional VLPs from baculovirus infected insect cells. Since these influenza VLPs are self-assembled influenza structural proteins and display similar functional and biochemical properties to wild-type influenza viruses, these influenza VLPs retain important structural conformations, including surface epitopes, necessary for an effective influenza vaccine.

Example 2

RT-PCR cloning of avian influenza A/hong Kong/1073/99 virus gene

It is an object of the present invention to provide synthetic nucleic acid sequences capable of directing the production of recombinant influenza virus proteins. Such synthetic nucleic acid sequences are obtained using reverse transcription and Polymerase Chain Reaction (PCR) methods using the viral native genomic RNA isolated from influenza virus. For the purposes of this application, nucleic acid sequences refer to RNA, DNA, cDNA or any synthetic variant thereof which encodes the protein.

Avian influenza a/hong kong/1073/99 (H9N2) virus is supplied by doctor k.subbarao (Centers for disease Control, Atlanta, Ga., USA). Viral genomic RNA was isolated using acid phenol RNA extraction using Trizol LS reagent (Invitrogen, Carlsbad, calif. usa) under CDC level 3 biosafety (BSL3) precautions. The cDNA molecules of these viral RNAs were obtained by reverse transcription using MuLV reverse transcriptase (InVitrogen) and PCR using oligonucleotide primers specific for HA, NA and M1 proteins and Taq I DNA polymerase (InVitrogen) (table 1). The PCR fragment was cloned between EcoRI sites in the bacterial subcloning vector pCR2.1TOPO (InVitrogen) to yield three recombinant plasmids containing HA, NA and M1cDNA clones.

Example 3

RT-PCR cloning of human influenza A/Sydney/5/94 (H3N2) virus genes

Influenza a/sydney/5/94 (H3N2) virus is supplied by m.massare doctor (Novavax, inc., Rockville, Md.). Viral genomic RNA was isolated using acid phenol RNA extraction using Trizol LS reagent (Invitrogen) under the BSL2 precaution conditions of novavax.inc. cDNA molecules of these viral RNAs were obtained by reverse transcription and PCR using oligonucleotide primers specific for HA, NA, M1, M2 and NP proteins (table 1). The PCR fragments were cloned between EcoRI sites in the bacterial subcloning vector pCR2.1TOPO, resulting in five recombinant plasmids containing HA, NA, M1, M2 and NPcDNA clones.

Example 4

Cloning of avian influenza A/hong Kong/1073/99 virus cDNA into baculovirus transfer vector

The HA, NA or M1 genes were subcloned from the pcr2.1topo based plasmid into the pFastBac1 baculovirus transfer vector (InVitrogen) within the polyhedrin locus and in the Tn7att site and downstream of the baculovirus polyhedrin promoter and upstream of the polyadenylation signal sequence. These viral genes were ligated using T4DNA ligase. For the HA gene, a BamHI-Kpn I DNA fragment from pCR2.1TOPO-HA was inserted into the plasmid DNA of pFastBac1 digested with BamHI-KpnI. For the NA gene, an EcoRI DNA fragment from pCR2.1TOPO-NA was inserted into plasmid DNA of pFastBac1 digested with EcoRI. For the M1 gene, an EcoRI DNA fragment from pCR2.1TOPO-M1 was inserted into the plasmid DNA pFastBac1 digested with EcoRI. Competent E.coli DH 5. alpha. bacteria (InVitrogen) were transformed with these DNA ligation reactions to generate transformed colonies, and bacterial clones were isolated. For the resulting pFastBac 1-based plasmid: pFastBac1-HA, pFastBac1-NA and pFastBac1-M1 were characterized by restriction enzyme mapping on agarose gels (FIG. 4 (A)). The nucleotide sequence of the cloned gene was determined by automated DNA sequencing, as shown in FIGS. 1-3. DNA sequence analysis showed that the cloned influenza HA, NA and M1 genes were identical to the nucleotide sequences of these genes previously disclosed [ influenza a/hong kong/1073/99 (H9N2) NA, HA and M1 genes (GenBank accession numbers AJ404629, AJ404626 and AJ278646, respectively) ].

Example 5

Cloning of human influenza A/Sydney/5/97 virus cDNA into baculovirus transfer vector

From the pCR2.1TOPO-based plasmid, HA, NA, M1, M2 and NP genes were subcloned into the pFastBac1 baculovirus transfer vector at the polyhedrin locus and in the Tn7att site downstream of the baculovirus polyhedrin promoter and upstream of the polyadenylation signal sequence. These viral genes were ligated using T4DNA ligase. For the HA gene, the BamHI-Kpn I DNA fragment from pCR2.1TOPO-hHA3 was inserted into the plasmid DNA pFastBac1 digested with BamHI-KpnI. For the NA gene, the EcoRI DNA fragment from pCR2.1TOPO-hNA was inserted into the plasmid DNA pFastBac1 digested with EcoRI. For the M1 gene, an EcoRI DNA fragment from pCR2.1TOPO-hM1 was inserted into the plasmid DNA pFastBac1 digested with EcoRI. For the M2 gene, an EcoRI DNA fragment from pCR2.1TOPO-hM2 was inserted into the plasmid DNA pFastBac1 digested with EcoRI. For the NP gene, the EcoRI DNA fragment from pCR2.1TOPO-hNP was inserted into plasmid DNA of pFastBac1 digested with EcoRI. Competent E.coli DH 5. alpha. bacteria (InVitrogen) were transformed with these DNA ligation reactions to generate transformed colonies, and bacterial clones were isolated. For the resulting pFastBac 1-based plasmid: pFastBac1-hHA3, pFastBac1-hNA2, pFastBac1-hM1, pFastBac1-hM2 and pFastBac1-hNP were characterized by restriction enzyme mapping on agarose gels. The nucleotide sequence of the cloned gene was determined by automated DNA sequencing. DNA sequence analysis showed that the cloned influenza HA, NA, M1, M2, and NP genes were identical in nucleotide sequence to those previously disclosed.

Example 6

Construction of a Polygenic baculovirus transfer vector encoding multiple avian influenza A/hong Kong/1073/99 Virus genes

To construct a pFastBac 1-based bacmid transfer vector expressing multiple influenza a/hong kong/1073/99 (H9N2) viral genes, a Sna BI-Hpa I DNA fragment containing the M1 gene from pFastBac1-M1 plasmid was first inserted into the Hpa I site of pFastBac 1-HA. This resulted in a pFastBac1-HAM plasmid, which simultaneously encodes HA and M1 in separate expression cassettes and is expressed under the control of different polyhedrin promoters.

Finally, the SnaBI-AvrII DNA fragment containing the HA and M1 expression cassette from pFastBac1-HAM was transferred into Hpa I-Avr II digested pFastBac1-NA plasmid DNA. This resulted in plasmid pFastBac1-NAHAM, which encodes 3 independent expression cassettes for expression of influenza HA, NA and M1 genes and is expressed under the control of different polyhedrin promoters (fig. 4 (B)).

In another example, the H3 gene from pFASTBA AC1-hHA3 (see example 5) was cloned into pFASTBA AC1-NAHAM as a fourth influenza virus gene for expression and production of heterotypic influenza VLPs.

Example 7

Production of multigenic recombinant baculoviruses encoding the NA, HA and M1 genes of avian influenza A/hong Kong/1073/99 viruses in insect cells

The resulting multigene bacmid transfer vector, pFastBac1-NAHAM, was used to generate multigene recombinant baculoviruses encoding the NA, HA and M1 genes of avian influenza a/hong kong/1073/99 (H9N2) for expression in insect cells. Recombinant bacmid DNA was generated by site-specific recombination between pFastBac1-NAHAM DNA and the AcMNPC baculovirus genome contained in competent escherichia coli DH10BAC cells (InVitrogen) on the polyhedrin and Tn7att DNA sequences (fig. 4 (B)). Recombinant bacmid DNA was isolated by the miniprep plasmid DNA method and transfected into Sf-9s cells using cationic lipid CELLFECTIN(InVitrogen). Following transfection, recombinant baculovirus was isolated, plaque purified, and amplified in Sf-9S insect cells. Viral stocks were prepared in Sf-9S insect cells and their expression of the avian influenza virus HA, NA and M1 gene products was characterized. The resulting recombinant baculovirus was named bNAHAM-H9N 2.

Example 8

Expression of recombinant avian influenza A/hong kong/1073/99 protein in insect cells

Sf-9S insect cells maintained at 28 ℃ as suspension cultures in shake flasks with serum-free medium (HyQ SFM, HyClone, Ogden, Utah) at 2X10⁶The cell density per cell/ml was infected with the recombinant baculovirus bNAHAM-H9N2, with a multiplicity of infection (MOI) of 3 pfu/cell. Viral infection was carried out for 72 hours to allow for influenza protein expression. The expression of avian influenza A/hong Kong/1073/99 (H9N2) HA and M1 proteins in infected insect cells was confirmed by SDS-PAGE and Western immunoblot analysis. SDS-PAGE analysis was performed on 4-12% linear gradient NuPAGE gels (Invitrogen) under reducing and denaturing conditions. The primary antibodies used in the Western immunoblot analysis were polyclonal rabbit antiserum raised against avian influenza a/hong kong/1073/99 (H9N2) challenge obtained from CDC and monoclonal mouse antiserum against influenza M1 protein (Serotec, UK). The secondary antibody used for Western immunoblot analysis was alkaline phosphatase-conjugated goat IgG antiserum raised against rabbit or mouse IgG (H + L) (Kirkegaard and Perry Laboratories, Gaithersburg, Md., USA). The results of these analyses (fig. 5) indicate that HA and M1 proteins are expressed in the baculovirus-infected insect cells.

Example 9

Purification of recombinant avian influenza H9N2 virus-like particles and macromolecular protein complexes

Culture supernatants (200ml) were collected by low speed centrifugation from Sf-9S insect cells infected with recombinant baculovirus bNAHAM-H9N2, wherein the bNAHAM-H9N2 expressed avian influenza a/hong kong/1073/99 (H9N2) HA, NA and M1 gene products. The culture supernatant was clarified by centrifugation at 4 ℃ for 1 hour in a Sorval RC-5B ultracentrifuge using a GS-3 rotor at 10,000 Xg. The virus and VLPs were separated from the clarified culture supernatant by centrifugation in a SorvalOTD-65 ultracentrifuge using a Sorval TH-641 cradle rotor at 27,000rpm for 3 hours at 4 ℃. The virus pellet was resuspended in 1ml PBS (pH7.2), loaded onto a 20-60% (w/v) discontinuous sucrose step gradient, and resolved in a SorvalOTD-65 ultracentrifuge using a Sorval TH-641 rotor at 27,000rpm, 4 ℃ for 16 hours. Fractions (0.5ml) were collected from the top of the sucrose gradient.

The sucrose gradient fractions were analyzed for influenza proteins by SDS-PAGE and Western immunoblot analysis as described in example 6 above. As shown by Western blot analysis, HA and M1 proteins appeared in the same sucrose gradient fractions (fig. 6), suggesting that HA and M1 proteins are associated as macromolecular protein complexes. Furthermore, HA and M1 proteins were present throughout the fractions of the sucrose gradient, suggesting that these recombinant viral proteins are associated with macromolecular protein complexes of varying density and composition.

Example 10

Analysis of recombinant avian influenza H9N2 VLPs and proteins by gel filtration chromatography

Protein macromolecules, such as VLPs and monomeric proteins, migrate differently on gel filtration or size exclusion chromatography columns depending on their mass size and shape. To determine whether the recombinant influenza protein from the sucrose gradient fraction is a monomeric protein or a macromolecular protein complex such as a VLP, a chromatography column (7 mm. times.140 mm) with a resin bed volume of 14ml Sepharose CL-4B (Amersham) was prepared. The size exclusion column was equilibrated with PBS and the column was calibrated with blue dextran 2000, yellow dextran and vitamin B12(Amersham Pharmacia) with apparent molecular weights of 2,000,000, 20,000 and 1,357, respectively, to determine the water volume outside the column. Blue dextran 2000 was also eluted from the column in the outer water volume (6ml fraction). As expected, the recombinant influenza protein complexes eluted from the column in the outer water volume (6ml fractions). The result is a characteristic of high molecular weight macromolecular protein complexes such as VLPs. Viral proteins in the column fractions were detected by Western immunoblot analysis as described in example 6 above. The M1 protein was detected in the outer water volume fraction (fig. 7). As expected, the baculovirus protein was also in the outer water volume.

Example 11

Electron microscopy of recombinant influenza VLPs

To determine whether the macromolecular protein complex containing the recombinant avian influenza protein isolated on the sucrose gradient has a morphology similar to influenza virions, negatively stained samples were subjected to electron microscopy. The recombinant avian influenza a/hong kong/1073/99 (H9N2) protein complex was concentrated and purified from the culture supernatant by ultracentrifugation on a discontinuous sucrose gradient as described in example 7. Aliquots of these sucrose gradient fractions were treated with 2% glutaraldehyde in PBS, ph7.2, absorbed onto freshly discharged plastic/carbon coated mesh, and washed with distilled water. The samples were stained with 2% sodium phosphotungstate ph6.5 and observed using a transmission electron microscope (Phillips). Electron micrographs of negatively stained samples of recombinant avian influenza H9N2 protein complex from both sucrose gradient fractions showed spherical and rod-shaped particles from both sucrose gradient fractions (fig. 8). These particles have different sizes (60 and 80nm) and morphologies. Larger complexes of both particles were also detected, as well as rod-shaped particles (fig. 8). All observed protein complex structures exhibit spike-like processes similar to influenza virus HA and NA enveloped particles. Since the size and appearance of the 80nm particles are similar to wild-type influenza virus particles, these structures are likely to be enveloped influenza VLPs. The smaller particles of about 60nm may be subviral particles that are morphologically and structurally different from the VLPs described above.

Example 12

Analysis of functional properties of influenza proteins by coagulation assays

To determine whether purified influenza VLPs and proteins have functional activities characteristic of influenza viruses, such as clotting and neuraminidase activities, purified influenza VLPs and proteins were tested in a clotting assay and a neuraminidase assay.

For the clotting assay, a series of 2-fold dilutions of either sucrose gradient fractions containing influenza VLPs or positive control wild-type influenza a virus were prepared. Then, they were mixed with PBS (pH7.2) containing 0.6% guinea pig erythrocytes and incubated at 4 ℃ for 1to 16 hours. PBS was used as negative control. The degree of coagulation was visually observed, and the highest dilution of the fraction capable of agglutinating guinea pig erythrocytes was determined (fig. 9). The highest coagulation titers observed for purified influenza VLPs and proteins were 1:4000, higher than the titers shown for the wild-type influenza control of 1: 2000.

Example 13

Analysis of functional Properties of influenza proteins by neuraminidase assay

The amount of neuraminidase activity in the sucrose gradient fraction containing influenza VLPs was determined by neuraminidase assay. In this assay, NA (enzyme) acts on a substrate (fetuin) and releases sialic acid. The enzyme activity was stopped by addition of arsenite reagent. The amount of sialic acid released was determined chemically with thiobarbituric acid, which produced a pink colour proportional to the amount of free sialic acid. The amount of colour (chromophore) was measured spectrophotometrically at a wavelength of 594 nm. The data are shown in fig. 8, indicating that VLP-containing sucrose gradient fractions produced significant amounts of sialic acid, and that these fractions corresponded to those exhibiting clotting activity.

Example 14

Immunization of BALB/c mice with functional homotypic recombinant influenza H9N2 VLPs

Immunogenicity of recombinant influenza VLPs was determined by immunizing mice followed by Western blot analysis of the immune sera. Recombinant VLPs consisting of viral HA, NA and M1 proteins from avian influenza a/hong kong/1073/99 (H9N2) and which had been purified by sucrose gradient were subcutaneously inoculated in the deltoid region of ten (10) female BALB/c mice on days 0 and 28 (1 μ g/injection). Five (5) mice were similarly administered PBS (pH7.2) as a negative control. Blood was drawn from the supraorbital cavity of the mice on day-1 (pre-bleed), day 27 (primary bleed) and day 54 (secondary bleed). Serum was collected from the blood samples by overnight clotting and centrifugation.

For Western blot analysis, 200ng of inactivated avian influenza A H9N2 or cold adapted avian influenza A H9N2 and protein standard (InVitrogen) pre-stained with See Blue Plus2 were taken, denatured (95 ℃,5 min), and electrophoresed at 172 volts in MES buffer on 4-12% polyacrylamide gradient NuPAGE gel (InVitrogen) under reducing conditions (10 mM. beta. -mercaptoethanol) until the bromophenol Blue track dye disappeared. For protein gels, electrophoretic proteins were visualized by staining with colloidal coomassie blue reagent (InVitrogen). Proteins were transferred from the gel to nitrocellulose membrane in methanol by standard Western blotting procedures. Serum from mice immunized with VLP and serum from rabbits with inactivated avian influenza virus H9N2 (positive control serum) were diluted 1:25 and 1:100, respectively, in PBS solution (pH7.2) for use as primary antibodies. The protein-bound membranes were blocked with 5% casein and then reacted with the primary antiserum at room temperature for 60 minutes with constant shaking. After washing the primary antibody membrane with phosphate buffered saline containing Tween20, the membrane was reacted with secondary antiserum [ goat anti-mouse IgG-alkaline phosphatase conjugate (1:10,000), or goat anti-rabbit IgG-alkaline phosphatase conjugate (1:10,000) ] for 60 minutes. After washing the second antibody membrane with phosphate buffered saline containing Tween20, the membrane bound antibody proteins are visualized by chromogenic substrates such as NBT/BCIP (InVitrogen).

The results of the Western blot analysis (figure 12) are: proteins with molecular weights similar to the viral HA and M1 proteins (75 and 30kd, respectively) were bound to positive control sera ((b) in fig. 12) and sera from mice immunized with recombinant influenza H9N2 VLPs ((a) in fig. 12). These results indicate that recombinant influenza H9N2 VLPs alone are immunogenic in mice by this route of administration.

Example 15

Hong Kong/1073/99 (H9N2) VLP immunogenicity and challenge studies in BALB/c mice

BALB/c mice were immunized on day 0 and day 21 with H9N2 VLPs (1. mu.g HA or 10. mu.g HA/dose) with or without 100. mu.g Novosome adjuvant and challenged with the homologous infectious virus IN on day 57. Mice were bled on days 0, 27 and 57, and sera were assayed for anti-HA antibodies using a Hemagglutination Inhibition (HI) assay with turkey red blood cells and for influenza by ELISA. The results of this study are shown in fig. 13-16.

High titers of H9N2 antibody were induced after a single immunization (priming) with H9N2 VLPs with or without Novasome and one dose of 10 μ g VLPs with 1 μ g HA (fig. 13). After one booster immunization, specific antibody titers increased by half to a logarithmic scale.

After immunization and booster immunization with 1 μ g HA in the form of H9N2 VLPs, serum HI levels reached or were higher than what was normally considered positive (log2=5) in all animals (fig. 14, bottom left panel). H9N2 VLPs co-formulated with Novasome adjuvant increased the HI response about 2-fold after primary immunization and about 4-fold after booster immunization (fig. 14, bottom right panel). The purified subunit H9N2 hemagglutinin also induced protective levels of HI antibody after booster immunization, whereas Novasome likewise increased the HI antibody response about 2-fold after primary immunization and about 4-fold after booster immunization (fig. 14, top panel). The level of HI antibodies induced by 10 μ g HA given as a subunit vaccine was equal to 1 μ g HA provided as VLP.

Furthermore, there was significantly less weight loss in H9N2 VLPs or VLP plus adjuvant vaccinated mice compared to unvaccinated control animals (fig. 15). There was no statistical difference in weight loss between the H9N2VLP vaccinated group and the H9N2VLP plus Novasome adjuvant vaccinated group.

Also, in mice immunized with H9N2 VLPs, pneumovirus titers at 3 and 5 days post challenge with H9N2 virus were significantly reduced (fig. 16). Addition of H9N2 VLPs to non-immunized control mice compared to mice immunized with VLPs onlyThe immunized mice had a significantly greater reduction in viral titer at day 3 (when influenza virus titer peaked in lung tissue).

Example 16

Type A/Fujian/411/2002 (H3N2) VLP immunogenicity and cross-reactivity between several influenza virus strains

BALB/c mice were immunized twice with type A/Fujian/411/2002 VLP (3.0, 0.6, 0.12, and 0.24 μ g HA/dose) Intramuscularly (IM) and Intranasally (IN). Mice were bled on day 0 and day 35. For serum, anti-HA antibodies were detected using a hemagglutination inhibition assay (HI) with turkey red blood cells and anti-influenza antibodies were detected by ELISA. The results of this study are shown in fig. 17A, 17B and 17C. These results indicate that immune responses against HA and NA are elicited both intramuscularly and intranasally.

Example 17

Determination of IgG isotypes in mice after vaccination with H3N2 VLPs

Mice were inoculated intramuscularly and intranasally with VLPs. Sera were taken at week 5 and analyzed to differentiate IgG isotypes.

Sera were tested on plates coated with purified ha (protein sciences) form a/wyoming/3/2003 using an ELISA assay system. Serial 5-fold dilutions of serum were added to the wells and the plates incubated. Biotinylated goat anti-mouse Ig, or anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, and anti-mouse IgG3 were then added. Streptavidin-peroxidase was then added to the wells. Detecting the bound conjugate. The results are shown in FIGS. 18A and 18B. These results indicate that IgG2a is the most abundant isotype in the anti-VLP immune response in mice.

Example 18

Type A/hong Kong/1073/99 (H9N2) VLP dose range study in SD rats

SD rats (each dose N ═ 6) were inoculated with purified a/hong kong/1073/99 (H9N2) VLPs (diluted to 0.12,0.6, 3.0 and 15.0 μ g HA with pH neutral PBS) or PBS alone on days 0 and 21. Blood samples were taken from the animals at day 0, day 21, day 35 and day 49 and the sera were subjected to a coagulation inhibition assay (HI) to detect functional antibodies capable of inhibiting the binding function of HA. The dose was based on HA content as determined by SDS-PAGE and scan density measurements of purified H9N2 VLPs. The results of the coagulation inhibition assay titers are shown in figure 19. A single 0.6 μ g HA dose of H9N2 VLPs or two doses of 0.12 μ g HA produced protective levels of HI antibodies in rats. These results indicate that lower amounts of HA can induce a protective response when HA is part of the VLP.

Example 19

Hong Kong/1073/99 (H9N2) VLP immunogenicity

BALB/C mice (0.12,0.6 μ g HA/dose) were immunized with H9N2 VLPs with or without 100 μ g Novosome and alum adjuvant on days 0 and 21 and challenged intranasally with homologous infectious virus on day 57. Mice were also immunized with 3.0 and 15.0 μ g HA/dose (without adjuvant). Mice were bled on days 0, 21, 35 and 49, and anti-HA antibodies were analyzed for serum using a hemagglutination inhibition assay (HI) with turkey red blood cells, and influenza was analyzed by ELISA. The results of this study are shown in fig. 20A and 20B.

These results indicate that a more robust overall immune response is observed when VLPs are administered with an adjuvant. However, a protective response was induced at week 3 with 0.12 μ g HA/dose compared to alum-containing VLP formulations and non-adjuvanted VLPs. Also at week 7, the HI potency of Novasomes containing VLPs increased by approximately 2 log compared to alum containing VLPs. The robustness of the response was similar to VLPs administered at 3.0 and 15.0 μ g HA/dose without adjuvant. These results indicate that Novasomes elicited responses that were more robust than alum. Furthermore, when VLPs are administered in Novasomes-containing formulations, a protective immune response can be achieved using 25-fold less of the VLPs.

Furthermore, the Novasomes formulation responded approximately 1.5 log higher than alum in the 0.6 μ g HA/dose data. The immune response was similar in magnitude to VLPs administered at 3.0 and 15.0 μ g HA/dose without adjuvant. These results indicate that approximately 5 times less VLP needs to be administered to achieve a protective response with the use of an adjuvant.

Furthermore, figure 20B shows HI titers of H9N2 VLPs using different Novasomes formulations. The following are the formulations used in the experiments:

group 1: H9N2VLP (0.1 μ g) (N =5)

Group 2: H9N2VLP (0.1. mu.g) DCW pure (N =5)

Group 3: H9N2VLP (0.1 μ g) has DCW1:3 (N =5)

Group 4: H9N2VLP (0.1 μ g) has DCW1:9 (N =5)

Group 5: H9N2VLP (0.1 μ g) has DCW1:27 (N =5)

Group 6: H9N2VLP (0.1 μ g) has NVAX1 (N =5)

Group 7: H9N2VLP (0.1 μ g) has NVAX 2(N =5)

Group 8: H9N2VLP (0.1 μ g) has NVAX3 (N =5)

Group 9: H9N2VLP (0.1 μ g) has NVAX4 (N =5)

Group 10: H9N2VLP (0.1 μ g) has NVAX5 (N =5)

Group 11: H9N2VLP (0.1 μ g) has Alum-OH (N =5)

Group 12: H9N2VLP (0.1 μ g) with CpG) (N =5)

Group 13: PBS (0.6. mu.g) (n =5)

Group 14: h3VLP (0.6 μ g) (n =5)

Group 15: h5VLP (0.6 μ g) (n =8)

-H9 (batch No. 11005)

Novasomes (batch No. 121505-2, polyoxyethylene-2-hexadecyl ether, cholesterol, ultrapure soybean oil, and cetylpyridinium chloride))

NVAX1: B35P83, MF-59replica (squalene, polysorbate, and Span)

NVAX2: B35P87 (Soybean oil, Brij, Cholesterol, Pluronic F-68)

NVAX3: B35P88 (Soybean oil, Brij, Cholesterol, Pluronic F-68, and polyethyleneimine)

NVAX4: B31P60 (squalene, Brij, cholesterol, oleic acid)

NVAX5: B31P63 (Soybean oil, Glycerol monostearate, Cholesterol, polysorbate)

-CpG (batch number 1026004)

-H5 (batch No. 22406)

Fig. 21 shows the dose response curve of H9N2 VLPs. The data indicate that the VLP dose of 0.6 μ g HA/dose is the minimum dose that elicits a protective immune response in mice after 3 weeks.

Example 20

Materials and methods for ferret research

Ferrets are purchased from Triple F Farms (FFF, Sayre, Pa.). All ferrets purchased had HAI titers of less than 10 coagulation units. Approximately 2 days prior to immunization, the animals were implanted with temperature transponders (BioMedic Data Systems, Inc.). Animals (6 animals/group) were vaccinated on day 0: (1) PBS (negative control, group 1); (2) influenza VLP H3N2 in an amount of 15 μ g H3 (group 2); (3) influenza VLP H3N2 in an amount of 3 μ g H3 (group 2); (4) influenza VLP H3N2 in an amount of 0.6 μ g H3 (group 3); (5) influenza VLP H3N2 in an amount of 0.12 μ g H3 (group 5); or (6) 15. mu.g of rH3HA (group 6). Animals were boosted with vaccine on day 21. Animals were bled on day 0 (prior to immunization), day 21 (prior to vaccine booster) and day 42. Animals were evaluated weekly for clinical signs of vaccine deleterious effects throughout the study period. Similar studies were performed with other influenza VLPs.

HAI levels in mink serum

Ferret serum was taken from FFF and tested for coagulation inhibition (HAI) following treatment with Receptor Destroying Enzyme (RDE) according to standard procedures (Kendal et al (1982)). In all ferrets selected for this study, the pre-existing antibody tests were negative for the human influenza virus in the epidemic (type a/new karlidonia/20/99 (H1N1), type a/panama/2007/99 (H3N2), type a/hulington/01/04 (H2N3) and type b/sichuan/379/99 and H5N1) (HAI ≦ 10).

Ferret

Approximately 8 months old, not exposed to influenza (influenza)) Male Fitch ferret (Mustella putorius furo) castrated and deodorized (spent) was purchased from FFF. Animals were housed in stainless steel rabbit cages (Shor-line, KS) containing Sani-chips Laboratory Animal Bedding (P.J. MurphyForest Products, N.J.). Ferrets are provided with Teklad universal Ferret Diet (Harlan Teklad, WI) and fresh water without limitation. Three dishes were changed weekly and cages were cleaned every two weeks.

Inoculation and blood sampling of ferrets

Vaccines [ e.g. influenza H3N2 VLPs or influenza H9N2 VLPs ] and controls [ e.g. rH3NA (a/wyoming/3/2003) and PBS (negative control) ] were stored at 4 ℃ prior to use. For most studies, 6 groups of ferrets were intramuscularly vaccinated with a volume of 0.5ml of vaccine concentrate or control (N-6/group).

Prior to blood collection and inoculation, animals were anesthetized by intramuscular injection of the medial thigh with a "KAX" solution of ketamine (25mg/kg), Atropine (0.05mg/kg), and Xylazine (2.0 mg/kg). Once the ferret is under anesthesia, it is placed in the supine position and blood (0.5-1.0 ml volume) is withdrawn from the anterior vena cava using a 23 "needle with a 1cc tuberculin syringe. The blood was transferred to a test tube containing a serum separator and a coagulation activator, and allowed to clot at room temperature. The tubes were centrifuged, serum removed and frozen at-80 ℃. Blood was collected before vaccination (day 0), booster (day 21) and day 42 and tested using the HAI assay.

Monitoring of ferrets

Body temperature was measured at approximately the same time each week throughout the study. The pre-inoculation values were averaged to obtain the baseline body temperature for each ferret. Changes in body temperature (in degrees fahrenheit) were calculated for each animal at each time point. Ferrets were examined weekly for clinical signs of vaccine deleterious effects including body temperature, weight loss, decreased liveness, runny nose, sneezing and diarrhea. Liveness levels were assessed using a scoring system based on the record of Reuman et al (1989), where 0 is alert, playful; 1 is alert, but only shows when stimulated; 2, the player is alert, but the player is not good to play when stimulated; 3, the player is not alert and does not have good playfulness when being stimulated. Based on the score of each animal in the group, the relative inactivity was calculated as: Σ (day 0 to day 42) [ activity score + 1]/∑ (day 0 to day 42), where n equals the total number of observations. A value of 1 is added to each base score so that the score "0" can be divided by the denominator to give an index value of 1.0.

Serum separation

Serum generally has low levels of non-specific coagulation inhibitors. To inactivate these non-specific inhibitors, the sera were treated with (RDE) before testing. Briefly, three RDEs were added to one serum and incubated at 37 ℃ overnight. The RDE was inactivated by incubation at 56 ℃ for about 30 minutes. After inactivation of the RDE, PBS was added to the sample to give a final serum dilution (RDE-Tx) of 1: 10. The diluted RDE-Tx serum was stored at 4 ℃ (7 days) or at-20 ℃ prior to testing.

Preparation of turkey red blood cells

Human influenza viruses bind sialic acid receptors containing N-acetylneuraminic acid alpha 2, 6-galactose linkages. Avian influenza viruses bind to sialic acid receptors containing N-acetylneuraminic acid α 2,3 galactose (α 2,3 linkage) and express α 2,3 and α 2,6 linkages. Turkey Red Blood Cells (TRBC) were used for HAI assay, since type a/fujian is a human influenza virus. TRBC was conditioned with PBS to obtain a 0.5% v/v suspension. Cells were stored at 4 ℃ and used within 72 hours after preparation.

HAI assay

The HAI assay is adapted from the CDC laboratory-based influenza surveillance handbook (Kendal et al (1982) definitions and procedures for laboratory based inflectionza surveillance, u.s.department of Health and Human Services, Public Health Service, Centers for disease Control, Atlanta, Georgia, the entire contents of which are incorporated herein by reference for all purposes). The RDE-Tx serum was serially diluted 2-fold in a v-bottomed microtiter plate. An equal volume of virus adjusted to approximately 8HAU/50ul was added to each well. The plates were incubated for 15 minutes at room temperature after capping and then 0.5% TRBC was added. The mixed plate was shaken, capped, and the TRBC allowed to settle at room temperature for 30 minutes. The HAI titer was determined from the reciprocal of the dilution of the last row containing unagglutinated TRBC. Each plate included positive and negative serum controls.

Example 21

A/hong Kong/1073/99 (H9N2) VLP dose range study in ferrets

Ferrets serologically negative for influenza virus as determined by coagulation inhibition were used to assess antibody and HI titers following vaccination with H9N2 VLPs. Ferrets were bled on days 0 and 21, and anti-HA antibodies were detected by the coagulation inhibition assay (HI) using turkey red blood cells on serum, and anti-influenza antibodies were detected by ELISA. The results are shown in FIG. 22. These results show HI titers corresponding to protective antibody levels at VLP doses of 1.5 and 15 μ g.

Example 21

Vaccination of H3N2 VLPs in ferrets

Ferrets were vaccinated with H3N2 VLPs of different virus strains at different doses (HA dose: 0.12,0.6, 3.0, 15.0 μ g) on day 0 and given boosts on day 21. The positive control was 15 μ g of rH3HA and the negative control was PBS alone. Sera were taken from ferrets on day 0 (pre-vaccination), day 21 (pre-booster), and day 42 as described above. HI assays were performed on serum samples to determine if an immune response to these VLPs was present. These data are shown in FIG. 23. These data indicate that H3N2 VLPs do elicit an immune response when introduced into ferrets. Thus, these H3N2 VLPs are immunogenic in ferrets.

Example 22

RT-PCR and cloning of HA, NA and M1 genes of influenza A/Indonesia/5/05 (H5N1) virus

Viral RNA of clade 2 influenza virus, type a/indonesia/5/05 (H5N1) strain was extracted using Trizol LS (Invitrogen, Carlsbad, CA) under BSL-3 precaution conditions. Reverse Transcription (RT) and PCR were performed on the extracted viral RNA using gene-specific oligonucleotide primers using a one-step RT-PCR system (Invitrogen). The following primer pairs were used for the synthesis of H5N1 Hemagglutinin (HA), Neuraminidase (NA) and matrix (M1) genes, respectively:

5’-AACGGTCCGATGGAGAAAATAGTGCTTCTTC-3' (SEQ ID.4) and

5’-AAAGCTTTTAAATGCAAATTCTGCATTGTAACG-3’(SEQ ID.5)(HA);

5’-AACGGTCCGATGAATCCAAATCAGAAGATAAT-3' (SEQ ID.6) and

5'-AAAGCTTCTACTTGTCAATGGTGAATGGCAAC-3' (SEQ ID.7) (NA), and

5’-AACGGTCCGATGAGTCTTCTAACCGAGGTC-3' (SEQ ID.8) and

5’-AAAGCTTTCACTTGAATCGCTGCATCTGCAC-3’(SEQ ID.9)(M1)

(ATG codon underlined).

After RT-PCR, cDNA fragments with molecular weights of 1.7, 1.4 and 0.7kB containing the influenza HA, NA and M1 genes, respectively, were cloned into pCR2.1-TOPO vector (Invitrogen). The nucleotide sequences of the HA, NA and M1 genes were determined by DNA sequencing. Clade 1H5N1 influenza virus was cloned from vietnam/1203/2003 according to a similar strategy.

Example 23

Generation of recombinant baculovirus containing H5N1

The HA gene was cloned as an RsrII-HindIII DNA fragment (1.7kb) downstream of the AcMNPV polyhedrin promoter in an RsrII and HindIII digested pFastBac1 bacmid transfer vector (Invitrogen). Similarly, the NA and M1 genes were cloned as EcoRI-HindIII DNA fragments (1.4 and 0.8kb, respectively) into EcoRI-HindIII digested pFastBac1 plasmid DNA. Three baculovirus transfer plasmids, pHA, pNA and pM1, containing influenza A/Indonesia/5/05 (H5N1) virus HA, NA and M1 genes, respectively, were obtained and used to generate recombinant bacmids.

After transformation of the influenza gene-containing bacmid transfer plasmid into escherichia coli DH10Bac competent cells (Invitrogen) containing AcMNPV baculovirus genome, bacmid was generated by site-specific homologous recombination. Recombinant bacmid DNA was transfected into Sf9 insect cells.

Nucleotide sequence of Indonesia/5/05 HA, NA and M1 gene

HA(SEQ ID NO:10)

NA(SEQ ID.11)

M1(SEQ ID.12)

One cloned HA gene, pHA5, contained two nucleotide changes nt #1172 and nt #1508 (in the wild type) compared to the wild type HA gene sequence. Using a similar strategy, clade 1H5N1 influenza virus was constructed and generated from vietnam/1203/2003 VLP (see below). The alignment of the nucleotide and amino acid sequences of pHA5 is as follows.

Alignment of hemagglutinin amino acid sequences

Example 26

Production of optimized influenza A/Indonesia/5/05 HA, NA and M1 genes expressed efficiently in Sf9 cells

The following polypeptides were derived from codon-optimized nucleotides corresponding to the Indonesia/5/05 HA gene (see example 31). These codon-optimized nucleotides were designed and prepared according to the methods disclosed in U.S. patent publication 2005/0118191 (the entire contents of which are incorporated herein by reference for all purposes) (Geneart GMBH, Regensburg, FRG). The nucleic acid sequence is shown in example 31.

Vac2-hac-opt (unmodified amino acid sequence) (SEQ ID27)

Vac2-hac-spc-opt (modified, signal peptide from chitinase, underlined) (SEQ ID28)

Vac2-hac-sph9-opt (modified, signal peptide from H9, underlined) (SEQ ID29)

Vac2-hac-cs-opt (-is a modified cleavage site) (SEQ ID30)

The following polypeptides correspond to unmodified, codon optimized NA and M1 genes that were also synthesized. Vac2-naj-opt (neuraminidase) (SEQ ID31)

Vac2-mc-opt (matrix) (SEQ ID32)

The synthetic codon-optimized HA, NA and M1 genes were subcloned into the pFastBac1 transfer vector using BamHI and HindIII sites as described above. Recombinant bacmid for expression of synthetic HA, NA and M1 genes in Sf9 cells was generated using e.coli DH10Bac strain (Invitrogen) as described above.

Example 24

RT-PCR cloning of clade 1 influenza A/Vietnam/1203/04 (H5N1)

The HA, NA and M1 genes were cloned by RT-PCR according to the method described above. The following sequence is a comparison of the disclosed genes with the cloned genes.

HA genes of clade 1 type A/Vietnam/1203/04 (H5N1)

And (3) a previous step: acc # AY818135HA gene (SEQ ID36)

The following steps: novavavax type A/Vietnam/1203/2004 (H5N1) HA gene (SEQ ID37)

Comparison of NA genes

Clade 1 type A/Vietnam/1203/04 (H5N1) NA gene (SEQ ID NO:39)

H5N1naLANL ISDN38704x NA_Viet1203_Lark(NVAX)(SEQ ID38)

Comparison of the M1 Gene

Clade 1 type A/Vietnam/1203/04 (H5N1) M1 gene (SEQ ID NO:40)

H5N1m1Lanl ISDN39958x M1_Viet1203_Lark(NVAX)(SEQ ID41)

All sequences were cloned and analyzed according to the methods disclosed above.

Example 25

Generation of optimized clade 1H5N1 influenza A/Vietnam/1203/04 HA, NA and M1 genes expressed efficiently in Sf9 cells

The following polypeptides were derived from codon-optimized nucleotides corresponding to type a/vietnam/1203/04. These nucleotides were designed and synthesized as disclosed above (see example 24) (Geneart GMBH, Regensburg, FRG).

VN1203-ha-cs-opt (modified cleavage site, underlined) (SEQ ID33)

VN1203-ha-spc-opt (modified signal peptide, underlined) (SEQ ID34)

VN1203-ha-sph9-opt (signal peptide and shaded cleavage site) (SEQ ID35)

Example 26

H5N1 Vietnam/1203/2003 VLP immunogenicity (limiting dose throttling)

BALB/C mice were immunized intramuscularly and intranasally with H5N1 VLPs at very low VLP doses (0.2, 0.04, 0.008, 0.0016 μ g HA/dose), and the mice were bled on days 0, 21 and 35. Mice were boosted on day 21. anti-HA antibodies were detected on sera using a hemagglutination inhibition assay (HI) with turkey red blood cells and influenza virus using ELISA. The results of this study are shown in figures 24 and 25.

These results indicate that a robust overall immune response is observed when VLPs are administered intramuscularly at very low doses. At 3.0 and 0.6 μ g HA/dose, the robustness of the response was similar to the control. These results show a true dose response, and an antibody response to 0.2 μ g VLPs of greater than 3.0 μ g rHA protein. Although the response upon intranasal administration was not as robust, a VLP dose of 0.2 μ g HA/dose did elicit robust responses. The ELISA titre at the 0.2. mu.g dose in this experiment was similar to the H3N2VLP vaccine at the 0.12. mu.g dose in the previous experiment, see above.

Example 27

Study of attacks

BALB/c mice were inoculated intramuscularly and intranasally with VLPs at concentrations of 3 μ g, 0.6 μ g, 0.12 μ g, and 0.02 μ g H3N2 VLPs (total HA dose), and then challenged with influenza A/Aichi/268 x 31. The results of this study are shown in figures 27 and 28. These results show that there was weight loss in all vaccinated animals, but animals vaccinated with 3.0 μ g and 0.12 μ g VLP recovered faster than other animals in both intramuscular and intranasal vaccinations. Intranasal dosage provides greater protection.

Example 29

Attack research (ferret)

In this study ferrets were vaccinated with H9N2 VLPs. A total of 18 ferrets were present in the challenge study: 6 received the simulant, 6 received the medium dose (1.5. mu.g) and 6 received the high dose (15.0. mu.g), all at intramuscular inoculation. Then, use 10⁶EID₅₀Type a/hong kong/1073/99 on ferret intranasal challenge. Nasal washes were collected on days 1,3, 5 and 7. Viral titers in nasal washes were determined on days 3, 5 and 7 for all animals. These data are shown in table 2 and fig. 29. These data show that by day 7, all vaccinated animals had no detectable virus in the nasal wash, while the mock group had detectable virus titers.

Table 2: wild type virus titer in ferrets after virus challenge (log10/ml)

Group (2): low dose

Group (2): high dose

Example 30

Intramuscular and intranasal vaccination studies in mice

Mice were inoculated intramuscularly or intranasally with 3 μ g, 0.6 μ g, 0.12 μ g or 0.024 μ g (total HA dose) concentrations of type a/foden/411/2002 (H3N2) VLPs on day 0 and boosted after 3 weeks. Control mice were inoculated with formalin inactivated wyomine a (fujian, vaccine strain) or PBS. Sera were collected from the vaccinated mice at weeks 0, 3, 5 and 8. The collected sera were analyzed for anti-HA antibodies by coagulation inhibition assay (HI) and anti-influenza antibodies by ELISA. The assay used influenza a/fujian/411/2002, a/panama/2007/99, a/wyoming/3/03 and a/new york/55/2004 strains of H3N 2. The results of this study are shown in FIGS. 30A-30H. These results indicate that H3N2 VLPs elicit antibodies against the parental influenza a/fujian/411/2002 strain and antibodies against other H3N2 strains. These results also indicate that the titer rose later in the intranasally vaccinated mice than in the intramuscularly vaccinated mice. However, after about 8 weeks, the intra-nasal titers were higher than the intra-muscular titers. Furthermore, after intramuscular vaccination, the titers against inactivated viral antigens appear to be similar to the equivalent dose of VLPs. However, inactivated antigens appear not to be as highly immunogenic after intranasal vaccination, and also not as broadly protective after intranasal vaccination.

Example 31

Production of optimized clade 2H5N1 influenza HA, NA and M1 genes for efficient expression in Sf9 cells

The following optimized nucleotides and polypeptides correspond to HA, NA and M1 of clade 2H5N1 virus type a/indonesia/5/05, type a/bar headed goose/qinghai/1A/2005 and type a/anhui/1/2005, which are designed and synthesized as disclosed above (Geneart GMBH, Regensburg, FRG). The optimized nucleotides and polypeptides are listed below. To make VLPs, type a/anhui HA can be expressed together with type a/indonesia NA and M1. To prepare VLPs containing type a/Qinghai HA and NA, the type a/indonesia M1 gene can be co-expressed with type a/Qinghai HA and NA.

Type A/Indonesia/5/05

Type A/Indonesia optimized HA (start and stop codons underlined) (SEQ ID42)

Type A/Indonesia HA protein sequence (SEQ ID NO:43)

Type A/Indonesia optimized HA (deletion of cleavage site)

(initiation and termination codons are underlined) (SEQ ID NO:44)

Type A/Indonesia HA protein sequence (SEQ ID NO:45)

Type A/Indonesia optimized NA (start and stop codons underlined)

(SEQ ID46)

Type A/Indonesia NA protein sequence (SEQ ID47)

Type A/Indonesia optimized M1(SEQ ID48)

Type A/Indonesia M1 protein sequence (SEQ ID49)

A type/Anhui/1/2005

Type A/Anhui optimized HA (start and stop codons underlined) (SEQ ID50)

Type A/Anhui HA protein sequence (SEQ ID51)

Type A/bar goose/Qinghai/1A/2005

Type A/Qinghai optimized HA (start and stop codons underlined) (SEQ ID52)

Type A/Qinghai HA protein sequence (SEQ ID53)

Type A/Qinghai optimized NA (start and stop codons underlined) (SEQ ID54)

Protein sequence:

type A/Qinghai NA protein sequence (SEQ ID55)

The following references are incorporated herein by reference:

Berglund,P.,Fleeton,M.N.,Smerdou,C.,and Liljestrom,P.(1999).Immunization with recombinant Semliki Forest virus induces protection againstinfluenza challenge in mice.Vaccine17,497-507.

Cox,J.C.,and Coulter,A.R.(1997).Adjuvants--a classification and review oftheir modes of action.Vaccine15,248-256.

Crawford,J.,Wilkinson,B.,Vosnesensky,A.,Smith,G.,Garcia,M.,Stone,H.,and Perdue,M.L.(1999).Baculovirus-derived hemagglutinin vaccines protectagainst lethal influenza infections by avian H5and H7subtypes.Vaccine17,2265-2274.

Crowther R A,Kiselev N A,Bottcher B,Berriman J A,Borisova G P,Ose V,Pumpens P.(1994).Three-dimensional structure of hepatitis B virus coreparticles determined by electron cryomicroscopy.Cell17,943-50.

Gomez-Puertas,P.,Mena,I.,Castillo,M.,Vivo,A.,Perez-Pastrana,E.,andPortela,A.(1999).Efficient formation of influenza virus-like particles:dependence on the expression levels of viral proteins.J.Gen.Virol.80,1635-1645.

Johansson,B.E.(1999).Immunization with influenza A virus hemagglutinin andneuraminidase produced in recombinant baculovirus results in a balanced andbroadened immune response superior to conventional vaccine.Vaccine17,2073-2080.

Lakey,D.L.,Treanor,J.J.,Betts,B.F.,Smith,G.E.,Thompson,J.,Sannella,E.,Reed,G.,Wilkinson,B.E.,and Wright,P.E.(1996)Recombinantbaculovirus influenza A hemagglutinin vaccines are well tolerated andimmunogenic in healthy adults.J.Infect.Dis.174,838-841.

Latham,T.,and Galarza,J.M.(2001).Formation of wild-type and chimericinfluenza virus-like particles following simultaneous expression of only fourstructural proteins.J.Virol.75,6154-6165.

Mena,I.,Vivo,A.,Perez,E.,and Portela,A(1996).Rescue of a syntheticchloramphenicol acetyltransferase RNA into influenza-like particles obtainedfrom recombinant plasmids.J.Virol.70,5016-5024.

Murphy,B.R.,and Webster,R.G.(1996).Orthomyxoviruses.In"Virology"(D.M.K.B.N.Fields,P.M.Howley,Eds.)Vol.1,pp.1397-1445.Lippincott-Raven,Philadelphia.

Neumann,G.,Watanabe,T.,and Kawaoka,Y.(2000).Plasmid-driven formationof influenza virus-like particles.J.Virol.74,547-551.

Olsen,C.W.,McGregor,M.W.,Dybdahl-Sissoko,N.,Schram,B.R.,Nelson,K.M.,Lunn,D.P.,Macklin,M.D.,and Swain,W.F.(1997).Immunogenicity andefficacy of baculovirus-expressed and DNA-based equine influenza virushemagglutinin vaccines in mice.Vaccine15,1149-1156.

Peiris,J.S.,Guan,Y.,Markwell,D.,Ghose,P.,Webster,R.G.,and Shortridge,K.F.(2001).Cocirculation of avian H9N2and contemporary"human"H3N2influenza A viruses in pigs in southwestern China:potential for geneticreassortmentJ.Virol.75,9679-9686.

Pumpens,P.,and Grens,E.(2003).Artificial genes for chimeric virus-likeparticles.In:"Artificial DNA"(Khudyakov,Y.E,and Fields,H.A.,Eds.)pp.249-327.CRC Press,New York.

Pushko,P.,Parker,M.,Ludwig,G.V.,Davis,N.L.,Johnston,R.E.,and Smith,J.F.(1997).Replicon-helper systems from attenuated Venezuelan equineencephalitis virus:expression of heterologous genes in vitro and immunizationagainst heterologous pathogens in vivo.Virology239,389-401.

Slepushkin,V.A.,Katz,J.M.,Black,R.A.,Gamble,W.C.,Rota,P.A.,and Cox,N.J.(1995).Protection of mice against influenza A virus challenged byvaccination with baculovirus-expressed M2protein.Vaccine13,1399-1402.

Treanor,J.J.,Betts,R.F.,Smith,G.E.,Anderson,E.L.,Hackett,C.S.,Wilkinson,B.E.,Belshe,R.B.,and Powers,D.C.(1996).Evaluation of arecombinant hemagglutinin expressed in insect cells as an influenza vaccine inyoung and elderly adults.J.Infect.Dis.173,1467-1470.

Tsuji,M.,et al.(1998).Recombinant Sindbis viruses expressing a cytotoxicT-lymphocyte epitope of a malaria parasite or of influenza virus elicit protectionagainst the corresponding pathogen in mice.J.Virol.72,6907-6910.

Ulmer,J.B.,et al.(1993).Heterologous protection against influenza by injectionof DNA encoding a viral protein.Science259,1745-1749.

Ulmer,J.B.,et al.(1998).Protective CD4+and CD8+T cells against influenzavirus induced by vaccination with nucleoprotein DNA.J.Virol.72,5648-5653.

Watanabe,T.,Watanabe,S.,Neumann,G.,and Kawaoka,Y.(2002)Immunogenicity and protective efficacy of replication-incompetent influenzavirus-like particles.J.Virol.76,767-773.

Zhou,X.,et al.(1995).Generation of cytotoxic and humoral immune responsesby non-replicative recombinant Semliki Forest virus.Proc.Natl.Acad.Sci.USA92,3009-3013.

other embodiments are as follows:

those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is intended that the appended claims cover such equivalents.

The present invention relates to the following items.

1. A virus-like particle (VLP) comprising influenza virus M1 protein and influenza viruses H5 and N1 hemagglutinin and neuraminidase proteins.

2. The VLP of item 1, wherein said M1 protein is derived from a different influenza virus strain than the H5 and N1 proteins.

3. The VLP of item 1, wherein said H5 or N1 is from a H5N1 clade 1 influenza virus.

4. The VLP of item 1, wherein said H5 and N1 are from H5N1 clade 2 influenza virus.

5. The VLP of item 3, wherein said H5 and N1 proteins comprise SEQ ID NOs 43 and 46, respectively, or sequences having at least 90% sequence identity to said sequences.

6. The VLP of item 4, wherein said H5 and N1 proteins comprise SEQ ID NOs 50 and 54, respectively, or sequences having at least 90% sequence identity to said sequences.

7. The VLP of item 1, wherein said H5 and N1 are derived from influenza virus isolated from an infected animal.

8. The VLP of item 7, wherein said infected animal is a human.

9. The VLP of item 1, wherein said VLP is expressed by a eukaryotic cell comprising one or more nucleic acids encoding influenza H5 and N1 proteins and influenza M1 protein under conditions that allow VLP formation.

10. The VLP of claim 9, wherein said eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, an amphibian cell, an avian cell, and a mammalian cell.

11. The VLP of item 10, wherein said eukaryotic cell is an insect cell.

12. The VLP of item 11, wherein said insect cell is Sf 9.

13. The VLP of item 1, wherein said VLP elicits neutralizing antibodies in a human or animal that are protective against influenza virus infection when administered to said human or animal.

14. An immunogenic composition comprising an effective dose of a VLP of any one of items 1-13.

15. The composition of item 14, wherein the composition comprises an adjuvant.

16. A vaccine comprising an effective dose of a VLP of any one of items 1-13.

17. The vaccine of item 16, wherein the vaccine comprises at least a second VLP comprising HA and NA from different influenza virus strains.

18. The vaccine of item 16 or 17, wherein the vaccine comprises an adjuvant.

19. The immunogenic composition or vaccine of items 15 or 18, wherein the adjuvant comprises

20. A method of inducing significant immunity to influenza virus infection in an animal comprising administering at least one effective dose of the vaccine of any one of items 16-18.

21. The method of item 20, wherein the vaccine is administered to the animal orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

22. The method of item 20 or 21, wherein the animal is a human.

23. Use of a VLP of any one of items 1-13 for the preparation of a vaccine for an animal, wherein said vaccine induces significant immunity to influenza virus infection in said animal.

24. The use of item 23, wherein the vaccine is administered to the animal orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

25. A method of making the VLP of any one of items 1-13, comprising expressing the M1, HA and NA proteins in a eukaryotic cell.

26. The method of item 25, wherein said eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, an amphibian cell, an avian cell, and a mammalian cell.

27. The method of item 26, wherein said eukaryotic cell is an insect cell.

28. The method of item 27, wherein said insect cell is Sf 9.

29. An insect cell expression vector encoding at least one influenza H5 or N1 protein.

30. A vaccine comprising an influenza VLP, wherein said VLP comprises influenza M1, HA and NA proteins, wherein said vaccine induces significant immunity to influenza virus infection in a human.

31. A vaccine comprising an influenza VLP, wherein said VLP consists essentially of influenza M1, HA and NA proteins, wherein said vaccine induces significant immunity to influenza virus infection in a human.

32. A vaccine comprising an influenza VLP, wherein said VLP comprises an influenza protein selected from the group consisting of influenza M1, HA and NA proteins, wherein said vaccine induces significant immunity to influenza virus infection in a human.

33. A method of inducing significant immunity to influenza virus infection in a human comprising administering at least one effective dose of the vaccine of any one of items 30-32.

34. The method of item 33, wherein the vaccine is administered orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

35. Use of an influenza VLP, wherein said VLP comprises influenza M1, HA and NA proteins, for the preparation of a vaccine, wherein said vaccine induces significant immunity against influenza virus infection in a human.

36. Use of an influenza VLP, wherein said VLP consists essentially of influenza M1, HA and NA proteins, for the preparation of a vaccine, wherein said vaccine induces significant immunity against influenza virus infection in a human.

37. Use of an influenza VLP, wherein said VLP comprises an influenza protein selected from the group consisting of influenza M1, HA and NA proteins, for the preparation of a vaccine, wherein said vaccine induces significant immunity to influenza virus infection in a human.

38. The use of any one of items 35-37, wherein the vaccine is administered to a human orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

39. The vaccine of any one of claims 16-19 and 30-32, wherein the vaccine has been treated to inactivate baculovirus.

40. The vaccine of item 40, wherein the inactivation treatment comprises incubating the sample containing the VLPs in about 0.2% beta-propiolactone (BPL) at about 25 ℃ for about 3 hours.

Claims (8)

1. A virus-like particle (VLP) comprising influenza virus M1 protein and influenza viruses H5 and N1 hemagglutinin and neuraminidase proteins.

2. A vaccine comprising an effective dose of the VLP of claim 1.

3. A method of inducing significant immunity to influenza virus infection in an animal comprising administering at least one effective dose of the vaccine of claim 2.

4. Use of the VLP of claim 1 for the preparation of a vaccine for an animal, wherein said vaccine induces significant immunity against influenza virus infection in said animal.

5. A method of making the VLP of claim 1, comprising expressing said M1, HA and NA proteins in a eukaryotic cell.

6. A vaccine comprising an influenza VLP, wherein said VLP comprises influenza M1, HA and NA proteins, wherein said vaccine induces significant immunity to influenza virus infection in a human.

7. A method of inducing significant immunity to influenza virus infection in a human comprising administering at least one effective dose of the vaccine of claim 6.

8. Use of an influenza VLP, wherein said VLP comprises influenza M1, HA and NA proteins, for the preparation of a vaccine, wherein said vaccine induces significant immunity against influenza virus infection in a human.