JP2014505681A - Modified influenza hemagglutinin protein and use thereof - Google Patents

Abstract

  The present invention relates generally to modified influenza hemagglutinin (HA) proteins and methods for their production and use, including their use in immunogenic compositions such as vaccines for the treatment and / or prevention of influenza infection.

Description

  This application claims priority from US Provisional Patent Application No. 61 / 426,390, filed December 22, 2010, the entire contents of which are incorporated herein by reference for all purposes. Shall be.

TECHNICAL FIELD The present invention relates generally to modified influenza hemagglutinin (HA) proteins and methods for their production and use, including their use in immunogenic compositions such as vaccines for the treatment and / or prevention of influenza infection. .

Description of electronically submitted document files The contents of the electronically submitted document files are hereby incorporated by reference in their entirety: a computer-readable format of the sequence listing Copy (file name: NOVV_046_00US_SeqList_ST25.txt, date of recording: October 29, 2010, file size: 10 kilobytes).

  Influenza viruses are members of the Orthomyxoviridae family and 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 base Sex protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). HA, NA, M1, and M2 proteins are membrane bound while NP, PB1, PB2, PA, and NS2 are bound to nucleocapsids. HA protein is an envelope glycoprotein responsible for virus attachment to host cells and is a major source of immunodominant epitopes for virus neutralization and protective immunity. Therefore, the HA protein is considered an important component for prophylactic influenza vaccines due to its relatively strong immunogenicity.

  Currently approved vaccines incorporating HA include inactivated influenza A and B virus vaccines (eg, trivalent vaccines for parenteral administration). Commercially available influenza vaccines are whole virus (WV) or subvirion (SV) virus vaccines. WV vaccines contain intact inactivated virions, while SV vaccines are treated with solvent and contain almost all of the viral structural proteins and part of the viral envelope. In addition, the SV virus contains mainly aggregates of HA, NA, and NP proteins when treated with detergent and further purified.

  In addition to attenuated WV or SV influenza vaccines, including HA, several recombinant HA products have been developed as vaccine candidates. These approaches focus on the expression, production and purification of influenza HA proteins and include the expression of these proteins using insect cells infected with baculovirus.

  More recently, in an attempt to produce more effective vaccines, HA can be expressed in combination with one or more additional viral proteins (eg, influenza M1 and / or NA), and protein giants such as virus-like particles (VLP). Attention has been focused on the formation of molecular particles (Pushko et al, 2005, Vaccine 23: 5751-9). VLPs mimic the overall structure of virus particles without the need for containing potentially infectious materials. VLPs lack the viral DNA or RNA genome but retain the three-dimensional structure of a real virus.

  Currently used HA-containing influenza VLPs are well tolerated after prophylactic administration, but are effective in certain populations, particularly young, elderly, and individuals with chronic illness Suspicious has been shown. Therefore, there is a need to improve influenza VLP vaccines for use in these highly susceptible patient populations. We addressed this need by developing influenza HA proteins that induce enhanced immunogenicity, especially when expressed in the context of influenza VLPs.

  The inventors have observed that removal of one or more glycosylation sites from influenza hemagglutinin (HA) protein surprisingly improves the ability of the HA protein to induce an immune response. In addition, the inventors have shown that HA proteins that have been modified to remove one or more glycosylation sites still retain their conformation and, unexpectedly, in the context of influenza VLPs. Were observed to retain the ability to interact with other influenza virus proteins such as M1 and NA. Thus, when produced in an appropriate expression system, such glycosylation site modifications will generally improve the immunogenicity of HA-based influenza vaccines (eg, influenza VLP vaccines). In this way, this discovery further reduces the required amount of viral antigen used per dose of vaccine, while at the same time increasing the vaccine yield per batch of vaccine produced.

  Accordingly, in one aspect, the invention provides a HA protein that has been modified to remove one or more glycans from a position on a hemagglutinin (HA) protein (ie, glycosylation site). Such a modified HA protein can be expected to induce a stronger immune response against infection with influenza virus than its unmodified counterpart. The glycans selected for removal can be found in either the stem region or the globular head of the HA protein. In certain embodiments, one or more glucans that are targeted for removal are found within the receptor binding domain (RBD) of the globular head. In various embodiments described herein, the glycan removed is an N-linked glycan (ie, a glycan bound to the side chain nitrogen of asparagine or arginine).

  In one embodiment, the HA protein is modified by manipulating the HA protein to possess one or more amino acid mutations that remove one or more glycosylation sites from the HA protein. The one or more amino acid mutations that remove one or more glycosylation sites from the HA protein, in one embodiment, are one amino acid mutation, or two amino acid mutation, or three amino acid mutation, or four amino acid mutation, or 5 Amino acid mutation. In one embodiment, amino acid mutations are made in the stem region. In another embodiment, the amino acid mutation is made at the globular head. In yet another embodiment, amino acid mutations are made in both the stem region and the globular head region. In one embodiment, one or more of the amino acid mutations occur in a receptor binding domain (RBD) within the globular head of the modified HA protein. In further embodiments, all amino acid mutations occur in the RBD within the globular head of the modified HA protein. In certain embodiments, one or more of the amino acid mutations are made by changing the NX (T / S) (SEQ ID NO: 1) glycosylation consensus sequence in the HA protein. In the sequence, X is any amino acid and T / S is a threonine residue or a serine residue. In an exemplary embodiment, the amino acid mutation is located at an amino acid position corresponding to an amino acid residue selected from 170-172 and 181-183 of SEQ ID NO: 2.

  In another embodiment, the HA protein is modified to remove one or more glycans via enzymatic treatment. In one embodiment, the HA protein is modified to remove one or more glycans via treatment with endoglycosidase. In other embodiments, the HA protein is modified to remove one or more glycans via chemical treatment. In certain embodiments, one or more glycans are chemically removed by a hydrazine-like chemical reaction.

  The modified influenza virus HA protein of the present invention may be derived from an interpandemic (eg, perennial or seasonal) influenza strain, or the HA protein may be derived from a strain that can cause pandemic outbreaks. (I.e., influenza strains with new hemagglutinin compared to the currently spreading strain of hemagglutinin, or influenza strains or humans that are pathogenic to avian subjects and can be transmitted horizontally in the human population) Influenza strain). Depending on the particular season and the nature of the HA antigen contained in the vaccine, influenza virus HA may be derived from one or more of the following subtypes: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. In typical embodiments, the influenza virus HA is derived from the H1, H2, H3, H5, H7, or H9 subtype.

  In various embodiments described herein, the HA protein may be derived from an avian influenza virus strain. In one embodiment, the avian influenza virus strain is H5N1. In another embodiment, the avian influenza virus strain is H9N2. In yet another embodiment, the avian influenza virus strain is H7N3. In yet another embodiment, the avian influenza virus strain is H7N7.

  In another embodiment, the HA protein may be derived from a swine influenza virus strain. In one embodiment, the swine influenza virus strain is H1N1. In another embodiment, the swine influenza virus strain is H1N2. In yet another embodiment, the swine influenza virus strain is H3N2.

  In another embodiment, the HA protein may be derived from a human influenza virus strain. In one embodiment, the human influenza virus strain is H1N1. In another embodiment, the human influenza virus strain is a B strain.

  In further embodiments, the immunogenic composition may comprise more than one modified HA protein. For example, the immunogenic composition can include a modified HA protein from an avian influenza virus strain and a modified HA protein from a swine influenza virus strain.

  In one embodiment, the modified HA protein exhibits hemagglutinin activity. Such modified HA proteins retain the ability to bind and aggregate red blood cells (erythrocytes).

  In another aspect, the invention provides a virus-like particle (VLP) comprising one or more modified influenza HA proteins. Such VLPs containing modified influenza HA proteins can be expected to induce a stronger immune response against influenza viruses than their VLP counterparts containing unmodified influenza HA proteins. In one embodiment, the modified HA protein comprises one or more amino acid mutations that remove one or more glycosylation sites from said HA protein. In a further embodiment, one or more of the amino acid mutations occur within the receptor binding domain (RBD) of the modified HA protein. In certain embodiments, one or more of the amino acid mutations are made by changing the NX (T / S) (SEQ ID NO: 1) glycosylation consensus sequence in the HA protein. In the sequence, X is any amino acid and T / S is a threonine residue or a serine residue. In an exemplary embodiment, the amino acid mutation is located at an amino acid position corresponding to an amino acid residue selected from 170-172 and 181-183 of SEQ ID NO: 2.

  In various embodiments described herein, the VLPs of the invention may include additional influenza proteins, including but not limited to M1, M2, NA, NP, PB1, PB2, and NS2. In an exemplary embodiment, the VLP of the invention comprises M1, NA, and a modified HA protein. In one embodiment, the M1 protein comprises amino acid residue YKKL at amino acid positions corresponding to positions 100-103 of SEQ ID NO: 3. In another embodiment, the M1 protein is derived from an avian influenza virus strain. In yet another embodiment, the M1 protein is derived from an avian influenza virus strain selected from H5N1, H9N2, H7N3, and H7N7. In certain embodiments, the M1 protein is derived from the avian influenza virus strain A / Indonesia / 5/05 (H5N1). In a more specific embodiment, the M1 protein from avian influenza virus strain A / Indonesia / 5/05 (H5N1) comprises SEQ ID NO: 3. In another embodiment, the M1 protein is derived from a swine influenza virus strain. In yet another embodiment, the M1 protein is derived from a swine influenza virus strain selected from H1N1, H1N2, and H3N2. In a particular embodiment, the M1 protein is derived from swine influenza virus strain A / California / 04/09 (H1N1). In a more specific embodiment, the M1 protein from swine influenza virus strain A / California / 04/09 (H1N1) comprises SEQ ID NO: 4.

  The present invention also provides a method of inducing substantial immunity against influenza virus infection in a mammal susceptible to influenza infection, wherein one or more glycosylation sites have been modified to remove one or more glycosylation sites. There is provided a method comprising administering at least one effective dose of a vaccine comprising a plurality of HA proteins. In an exemplary embodiment, the present invention is a method for inducing substantial immunity against influenza virus infection in a mammal susceptible to influenza, such that one or more glycosylation sites are removed. There is provided a method comprising administering at least one effective dose of a vaccine comprising an influenza VLP comprising one or more modified HA proteins. In one embodiment, the mammal is a human. In one embodiment, the method comprises administering the vaccine to an animal orally, parenterally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. including.

  The present invention is also a method of formulating a vaccine that induces substantial immunity against influenza virus infection in a mammal susceptible to influenza infection, modified to remove one or more glycosylation sites. Also provided is a method comprising adding to the formulation an effective dose of a vaccine comprising one or more HA proteins. In an exemplary embodiment, the present invention provides a method of formulating a vaccine that induces substantial immunity against influenza virus infection in a mammal susceptible to influenza infection, comprising one or more glycosylation sites. Provided is a method comprising adding to the formulation an effective dose of a vaccine comprising an influenza VLP comprising one or more HA proteins modified for removal.

  In another aspect, the invention provides one or more recombinant constructs encoding an HA protein and at least one additional structural protein from influenza virus that have been modified to remove one or more glycosylation sites. To provide a method for producing influenza-derived VLPs. In an exemplary embodiment, the VLP comprises M1, NA, and an HA protein that has been modified to remove one or more glycosylation sites. In one embodiment, one or more recombinant constructs are used to transfect, infect, or transform a suitable host cell. In an exemplary embodiment, the recombinant construct is a baculovirus construct. The host cell is cultured under conditions that allow expression of the modified HA protein and at least one structural protein from influenza virus, and a VLP is formed in the host cell. Infected cell medium containing influenza VLPs is collected and the VLPs are purified. In various embodiments described herein, the host cell can be a eukaryotic cell, and in a typical embodiment, the host cell is an insect cell.

  The present invention also provides a progenitor containing an influenza VLP comprising one or more HA proteins modified to remove one or more glycosylation sites and at least one additional structural protein from influenza virus. Features a method of formulation. In one embodiment, one or more recombinant constructs encoding a modified HA protein and at least one structural protein from influenza virus are used to transfect, infect, or transform a suitable host cell. . In an exemplary embodiment, the recombinant construct is a baculovirus construct. The host cell is cultured under conditions that allow expression of the modified HA protein and at least one structural protein from influenza virus, and a VLP is formed in the host cell. The influenza VLP is isolated and purified, and the drug substance is formulated containing the influenza VLP. The drug substance may further contain an adjuvant. In addition, the present invention provides a method of formulating a drug product by mixing such a drug substance containing influenza VLPs with lipid vesicles, ie non-ionic lipid vesicles. Thus, influenza VLPs can bud as encapsulated particles from infected cells. Sprouting influenza VLPs are isolated and purified by ultracentrifugation or column chromatography as the bulk, and as drug products such as vaccines, alone or adjuvants such as Novovax, Inc., a product of Novavax, Inc. And can be formulated together. Novasomes® that enhance immunological effects are described in further detail in US Pat. No. 4,911,928, which is incorporated herein by reference.

It is a figure which shows the arrangement | sequence of A / Indonesia / G5 / 2005 (H5N1) HA1 arrangement | sequence. The glycan positions of amino acid residues 170-172 and 181-183 are illustrated. Modified to remove glycosylation sites at amino acid positions 170-172 (lanes 4-6), amino acid positions 181-183 (lane 2), or both amino acid positions 170-172 and 181-183 (lane 3) It is a figure which shows SDS-PAGE of A / Indonesia / 05/2005 (H5N1) influenza VLP containing the made HA protein.

Detailed description definition:
The term “antigen preparation” or “antigen composition” as used herein refers to a preparation that induces an immune response when administered to a vertebrate, particularly a bird or mammal.

  The term “adjuvant” as used herein is a compound that, when used in combination with a particular immunogen (eg, VLP) in a formulation, enhances or otherwise alters or modifies the resulting immune response. Point to. Modification of the immune response includes enhancement of either or both of the antibody and the cellular immune response or expansion of its specificity. Modification of the immune response may also mean a reduction or suppression of a particular antigen-specific immune response.

  The term “avian influenza virus” as used herein refers to an influenza virus that is found primarily in birds but can also infect humans or other animals. In some cases, the avian influenza virus can be transmitted or transmitted from person to person. Avian influenza viruses that infect humans can cause influenza pandemics, ie human morbidity and / or death. A pandemic occurs when a new strain of influenza virus (a virus that humans do not have innate immunity) emerges, spreads across individual locations, and possibly the entire globe, and infects many people at once.

  The term “chimeric protein” as used herein refers to a construct in which at least two heterologous proteins are linked to a single macromolecule (eg, a fusion protein).

  As used herein, an “effective dose” generally refers to induction of immunity, prevention and / or amelioration of influenza virus infection, or reduction of at least one symptom of influenza infection, and / or enhancement of VLP efficacy at additional doses. Refers to the amount of VLP of the present invention sufficient. An effective dose may refer to an amount of VLP sufficient to delay or minimize the onset of influenza infection. Effective dose may also refer to the amount of VLP that provides a therapeutic benefit in the treatment or management of influenza infection. Furthermore, an effective dose is that amount of a VLP of the invention that provides a therapeutic benefit in the treatment or management of influenza infection, alone or in combination with other therapies. An effective dose may also be an amount sufficient to enhance the subject's (eg, human) 's own immune response to subsequent exposure to influenza virus. The level of immunity can be monitored, for example, by measuring neutralizing secretion and / or the amount of serum antibodies, such as plaque neutralization, complement binding, enzyme-linked immunosorbent, or microneutralization experimental assays. In the case of a vaccine, an “effective dose” is an amount that prevents the disease or reduces the severity of symptoms.

  As used herein, the term “influenza VLP” refers to a VLP comprising at least one influenza protein. Said VLP may comprise additional influenza proteins and / or non-influenza proteins.

  The term “hemagglutinin” (or “HA” or “HA protein” or “hemagglutinin protein”) as used herein refers to a polypeptide whose amino acid sequence comprises at least one unique sequence of HA. A wide variety of HA sequences derived from influenza isolates are known in the art; in fact, the National Center for Biotechnology Information (NCBI) has more than 9000 HA sequences at the time of filing this application. A database (www.ncbi.nlm.nih.gov/genomes/FLU/flu.html) is included. One skilled in the art can refer to this database with reference to HA polypeptides in general and / or specific HA polypeptides (eg, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12). , H13, H14, H15, or H16 polypeptide; or a specific host such as avian, camel, dog, cat, civet, environment, horse, human, leopard, mink, mouse, seal, swallow, pig, Sequences unique to HA) that mediate infections such as tigers and whales can be readily identified.

  As used herein, the term “hemagglutinin activity” refers to the ability of HA-containing proteins, VLPs, or portions thereof, to bind and aggregate red blood cells (erythrocytes).

  As used herein, the terms “N-linked glycans” and “N-linked oligosaccharides” refer to oligosaccharides that are covalently attached to a conjugate (eg, HA) by N-glycosidic linkages.

  As used herein, the term “neuraminidase activity” refers to enzymatic activity in which NA-containing proteins, VLPs, or portions thereof cleave sialic acid residues from substrates including proteins such as fetuin.

  As used herein, the term “infectious agent” refers to a microorganism that causes infection in a vertebrate. Usually, the organism is a virus, bacterium, parasite, and / or fungus. The term also refers to different antigenic variants of the same infectious agent.

  As used herein, the term “immunostimulatory substance” refers to a compound that enhances the immune response through the body's own chemical mediator (cytokine). These molecules are interleukins (eg IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (eg granulocyte-macrophage (GM) -colony stimulation) Various cytokines having immunostimulatory, immunopotentiating and pro-inflammatory activities, such as macrophage inflammatory factor, Flt3 ligand, B7.1; other immunostimulatory molecules such as B7.2; Includes lymphokines and chemokines. The immunostimulatory molecule can be administered in the same formulation as the influenza VLP or can be administered separately. Either this protein or an expression vector encoding this protein can be administered to produce an immunostimulatory effect.

  The term “immunity” as used herein refers to the induction of the vertebrate immune system, which leads to the prevention, amelioration, and / or reduction of at least one infectious symptom of the vertebrate. Immunization may also refer to a hemagglutination inhibition (HI) titer of 40 or more when a VLP of the invention is administered to a vertebrate and the VLP induces an immune response against influenza virus HA.

  As used herein, the term “receptor binding domain” (or “RBD”) refers to an approximately 148 amino acid domain comprising the sialic acid binding site of HA. The subregion of the HA receptor binding domain, known as the RBD-A (“parietal”) domain, is based on the numbering established for HA from influenza strain A / South Carolina / 1/1918, Contained within amino acids 131-143, 170-182, 205-215, and 257-262 (Chih-Jen Wei et al, 2010, Set Transl Med 2: 24ra21).

  As used herein, the term “seasonal influenza virus” has spread throughout the human population during a given influenza season, based on epidemiological studies conducted by the National Influenza Centers around the world. Influenza virus strains determined to be Some of these epidemiological studies and isolated influenza viruses are based on four World Health Organization (WHO) reference laboratories, one of which is Centers for Disease Control and Prevention in Atlanta. for detailed testing in one of the Disease Control and Prevention (CDC). These laboratories test how well antibodies made against current vaccines respond to spreading viruses and new influenza viruses. This information is compiled with information on influenza activity and presented to the US Food and Drug Administration (FDA) Advisory Board and WHO meetings. These meetings will select three viruses (two subtypes of influenza A virus and one influenza B virus) to be included in the influenza vaccine for the next fall and winter. This selection is made in February for the Northern Hemisphere and in September for the Southern Hemisphere. Usually, one or two of the three virus strains in the vaccine are changed annually.

  As used herein, the term “swine influenza virus” refers to an influenza virus that is found primarily in pigs but can also infect humans or other animals. In some cases, swine influenza virus can be transmitted or transmitted from person to person. Swine influenza viruses that infect humans can cause an influenza pandemic, ie human morbidity and / or death. A pandemic occurs when a new strain of influenza virus (a virus that humans do not have innate immunity) emerges, spreads across individual locations, and possibly the entire globe, and infects many people at once.

  The term “vaccine” as used herein refers to a preparation of dead or weakened pathogens, or a preparation of derived antigenic determinants used to induce antibody formation or immunity against the pathogen. . Vaccines are administered to provide immunity against disease, eg, influenza caused by influenza virus. In addition, the term “vaccine” refers to a suspension of an immunogen (eg, VLP) administered to a vertebrate to provide protective immunity, ie, immunity that prevents or reduces the severity of the disease associated with the infection. Also refers to solution. The present invention provides a vaccine composition that is immunogenic and can provide a protective action against diseases associated with infection.

  As used herein, the term “vertebrate” or “subject” or “patient” includes, but is not limited to, humans and other non-human primates such as chimpanzees and other apes and monkeys. Refers to any member of the Chordate subgenus, including. Domestic animals such as cattle, sheep, pigs, goats, and horses; pet mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; chickens, turkeys, and other poultry birds, ducks Birds including pet birds, wild birds, and hunting birds, such as geese, are also non-limiting examples. The terms “mammal” and “animal” are included in this definition. It is intended to include both mature and newborn individuals.

  As used herein, the term “virus-like particle” (VLP) refers to a structure that is similar to a virus in at least one form but has not been demonstrated to be infectious. The virus-like particle according to the present invention does not retain the genetic information encoding the protein of the virus-like particle. In general, virus-like particles lack the viral genome and are therefore non-infectious. In addition, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.

Influenza hemagglutinin (HA):
Influenza virus is an RNA virus characterized by a lipid membrane envelope containing two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), embedded in the membrane of a viral particle. There are 16 known HA subtypes and 9 NA subtypes, and the various influenza strains are named based on the number of HA subtypes and NA subtypes of the strain. Based on a comparison of amino acid sequence identity and crystal structure, HA subtypes were classified into two main groups and four smaller clades. Different HA subtypes do not necessarily share strong amino acid sequence identity, but the overall three-dimensional structure of the different HA subtypes is similar to each other and some subtleties that can be used for classification purposes. There are significant differences. For example, the specific orientation of the membrane distal subdomain relative to the central α-helix is one of the structural characteristics commonly used to determine HA subtypes (Russell et al., Virology, 325: 287, 2004).

  HA is a homotrimeric integral membrane glycoprotein. HA is cylindrical and contains an ectodomain composed of a spherical head and a stem region. Both the globular head and stem regions of HA carry N-linked oligosaccharides that can affect the functional properties of this protein. For example, the HA mutants lacking one or more stem oligosaccharides show reduced cell fusion capacity, which concludes that the oligosaccharides in the stem region maintain the metastable form of HA required for cell fusion. (Ohuchi et al., 1997, J. Virology 71: 3719-25). Furthermore, the presence of glycans in the vicinity of the proteolytic active site of HA has been shown to regulate cleavage and affect influenza virus pathogenicity (Deshpande et al, 1987, Proc Natl Acad Sci USA 84: 36-40). Furthermore, glycosylation of influenza virus HA is important to protect against proteolysis (Schwartz et al., 1976, J. Virology 19: 782-91).

  Little is known about how glycan structure and composition affect HA activity, but the role of glycans in stabilizing HA conformation and the ability of HA to interact with other influenza proteins It has been suggested that glycans have an effect on the brain (Wilson et al, 1981, Nature 289: 373-8). Indeed, it has been found that the growth of viruses lacking one or more glycans from the globular head region is impaired in MDCK cells and embryonated chicken eggs (Wagner et al, 2001, Intl Cong). Series 1219: 375-82). This is mainly due to the limited release of offspring from host cells as a result of the enhanced receptor affinity of mutant HA. Id. Furthermore, when glycans were removed from the HA stem, the resulting virus showed temperate sensitive growth in cell culture and chicken eggs. These viruses have reduced pH stability, indicating that glucan is important to maintain the HA protein in the proper conformation.

Modified hemagglutinin (HA) protein:
We can produce influenza virus-like particles (VLPs) containing HA proteins lacking one or more glycans, and surprisingly retain the activity and potency of HA and NA I found out. Such results are unexpected when the glycan's claimed role in maintaining HA conformation and stability is specifically envisioned. Furthermore, modified HA proteins have enhanced immunogenicity when expressed alone or in the context of influenza VLPs compared to their unmodified counterparts. Thus, in one aspect, this application describes the generation of modified HA proteins with increased immunogenicity that may facilitate the production of improved vaccines.

  In accordance with the present invention, any number of modifications can be made to the HA protein that remove glycans from positions on the HA protein (ie, glycosylation sites), and in one embodiment an immune response against influenza virus infection To increase the ability to stimulate, multiple glycans (eg, 2, 3, 4, 5, or 6 glycans) can be removed. In an exemplary embodiment, the one or more glycans selected for removal are N-linked glycans. In a further embodiment, only N-linked glycans are removed. Such modifications can occur as a result of engineered point mutations, frameshift mutations, deletions or insertions, and can be one or more (eg, 1, 2, 3, 4, 5, 6, 7 The above point mutation is preferable. In another embodiment, one or more glycans can be removed via enzymatic or chemical treatment. For example, the HA protein can be modified to remove one or more N-linked glycans via endoglycosidase treatment. In other embodiments, N-linked glycans can be removed chemically by hydrazine-like chemistry.

  In one aspect, one or more glycosylation sites can be removed from the HA protein by mutating a nucleic acid sequence encoding the HA protein. Mutations to remove one or more glycans can be introduced into the HA proteins of the invention using any methodology known to those skilled in the art. For example, modified HA proteins can be made using oligonucleotide directed mutations that allow any possible type of base pair change at any determinant site along the encoded DNA molecule. In general, this approach involves annealing an oligonucleotide that is complementary (except for one or more mismatches) to a single-stranded nucleotide sequence encoding the HA protein of interest. This mismatched oligonucleotide is then extended by a DNA polymerase to produce a double-stranded DNA molecule containing the desired change in the sequence of one strand. For example, this change in sequence can result in amino acid deletions, substitutions, or insertions. The double-stranded polynucleotide can then be inserted into an appropriate expression vector, thus producing a mutated or modified polypeptide. The oligonucleotide directed mutation can be performed via PCR, for example.

  The HA protein used in the compositions and methods of the invention includes any HA protein that has the ability to stimulate an immunogenic response to influenza virus infection. Such HA proteins include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. As will be appreciated by those skilled in the art, HA proteins modified to remove glycosylation sites can be obtained by recombinant or genetic engineering techniques that are routine and well known in the art. The modified HA protein can be obtained, for example, by mutating a gene encoding the target HA protein by site-specific or random mutagenesis. Such mutations can include point mutations, deletion mutations, and insertion mutations. For example, one or more point mutations (eg, substitution of one or more amino acids with one or more different amino acids) can be used to construct the modified HA proteins of the invention.

  The present invention has been modified to remove one or more glycosylation sites to increase the ability to stimulate an immune response against influenza by 5%, 10% at the amino acid level with the wild-type HA protein, Homologous HA that is 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical It further includes a protein. The present invention also includes nucleic acid molecules that encode the HA proteins described above.

  The invention also includes one or more activities comprising at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 amino acid residues and associated with a modified HA protein A fragment of a modified HA protein that retains Such fragments are enzymatically digested with the HA protein of interest by deletion mutations using routine and well-known recombinant techniques in the art, or using any of several well-known proteolytic enzymes. Can be obtained. The present invention further includes nucleic acid molecules that encode the modified HA proteins and HA protein fragments described above.

  In one aspect, the target amino acid substitution is made at one or more glycosylation sites of the HA protein of interest. In one embodiment according to this aspect, one or more amino acids in the N-linked glycosylation consensus sequence of NX (T / S) are mutated to eliminate the glycosylation signal sequence. Such mutations generally prevent N-linked glycosylation and improve the immunogenicity of the HA protein.

  Methods for identifying the N-linked glycosylation site of the HA protein of interest are known in the art. For example, the NetNGlyc 1.0 server (Technical University of Denmark) can be used to predict the N-linked glycosylation site of NA. This software compares the sequence to be matched with a database of known N-linked glycosylation sites and presents a glycosylation possible score. A score of more than 0.5 indicates a site with a possibility of glycosylation, and a score of less than 0.5 indicates a less likely site. The NCBI protein database can then be searched using BLAST for putative glycosylation sites identified using NetNGlyc 1.0 software for matching sequences in native influenza viruses.

  In one embodiment, one or more glycosylation sites are removed from the globular head region. In another embodiment, one or more glycosylation sites are removed from the stem region. In yet another embodiment, one or more glycosylation sites are removed from both the globular head region and the stem region. In an exemplary embodiment, one or more glycosylation from the top of the globular head in a region known as the receptor binding domain (RBD), which is an approximately 148 amino acid domain carrying a sialic acid binding site. The site is removed. RBD, also known as receptor binding site (RBS), is at the distal end of the membrane of each HA monomer, and its specificity for sialic acid and the nature of its binding to adjacent galactose residues determine host range restrictions . In certain embodiments, one or more glycosylation sites are removed from a subregion of RBD known as RBD-A. RBD-A ("Parietal" domain) is based on the numbering established for HA from influenza strain A / South Carolina / 1/1918, amino acids 131-143, 170-182, 205-215, and Contained within 257-262 (GenBank accession number AF117241) (Chih-Jen Wei et aL., 2010, Sci. Transl Med 2: 24ra21).

  In one embodiment, amino acid substitutions are made with one or more NX (T / S) N-linked glycosylation consensus sequences in the RBD. In the sequence, X is any amino acid and (T / S) is either a threonine residue or a serine residue. In an exemplary embodiment, the one or more amino acid substitutions correspond to amino acid residues selected from 170-172 and 181-183 of the HA protein of A / Indonesia / 5/05 (SEQ ID NO: 2). Done in Thus, one or more of the natural amino acids at these positions are Ala, Asn, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Any other amino acid can be substituted, including Trp, Tyr, and Val. Specific examples of HA proteins showing an increased ability to stimulate an immune response against influenza virus infection include (1) threonine at position 172 replaced with alanine and / or (2) threonine at position 183 replaced with alanine HA protein. As will be appreciated by those skilled in the art who have mastered the disclosure of the present application, when amino acid mutations are made at positions corresponding to asparagine residues 170 and / or 181 of the HA protein (SEQ ID NO: 2) of A / Indonesia / 5/05 Similar results can be obtained.

  According to the present invention, one or more mutations can be made in any HA protein of interest to increase the ability of the HA protein to stimulate an immune response to influenza virus infection. Such mutations include point mutations, frameshift mutations, deletions and insertions. In one embodiment, one or more point mutations that result in one or more amino acid substitutions are used to produce an HA protein with an increased ability to stimulate an immune response against influenza virus infection. In an exemplary embodiment of the invention, the N170 position (eg N170D), T172 of the HA protein of A / Indonesia / 5/05 (SEQ ID NO: 2) is used to produce the desired result for other HA proteins of interest. One or more mutations can be made at positions equivalent to or corresponding to positions (eg, T172A), N181 (eg, N181D), and / or T183 (eg, T183A).

  Corresponding positions of the HA proteins identified herein (eg, the HA of A / Indonesia / 5/05 of SEQ ID NO: 2) can be readily identified for other HA proteins by those skilled in the art. Thus, given the designated regions and assays described in this application, one or several modifications that would increase the ability to stimulate an immune response against influenza virus infection in any HA protein of interest will be made by those skilled in the art. Can do.

  In preferred embodiments, the modified HA protein has one or more amino acid substitutions selected from positions corresponding to N170, T172, N181, and / or T183 compared to the wild-type HA protein. In other embodiments, the modified HA protein has additional amino acid substitutions at other positions as compared to the respective wild type HA protein. Thus, the modified HA protein may have at least about 2, 3, 4, 5, 6, 7 or more different residues at other positions as compared to the respective wild type HA. As one skilled in the art will recognize, the number of additional positions that may have amino acid substitutions will depend on the wild-type HA protein used to generate the variant. For example, HA from X31 influenza strain shows seven N-linked glycosylation sites (Ermonval et al, 2000, Glycobiology 10: 77-87). Thus, in some cases, as many as 7 or more different N-glycans can be removed via targeted amino acid substitution.

  As described above, in another embodiment, one or more glycans can be removed by enzymatic treatment. For example, the HA protein can be modified to remove one or more N-linked glycans via treatment with endoglycosidase. As used herein, “endoglycosidase” is used to refer to an enzyme or functional portion, derivative and / or analog thereof capable of significantly cleaving a sugar component from a substrate containing an oligosaccharide. . The use of such enzymes that release oligosaccharides from glycoproteins (eg, HA) or glycolipids is described in US Pat. No. 7,632,654, which is hereby incorporated by reference in its entirety. . Examples of endoglycosidases suitable for removal of N-glycans include endoglycosidases D, F, F1, F2, and H.

  In other embodiments, one or more N-linked glycans can be chemically removed. In one embodiment, the chemical reaction is mediated by one or more hydrazine-like reagents. Methods for N-linked glycan release using hydrazine and hydrazine-like reagents are found in US Pat. No. 5,539,090, which is hereby incorporated by reference in its entirety. Briefly, before subjecting HA to the action of hydrazine reagent (ie hydrazine degradation), preferably the HA protein and hydrazine reagent are prepared as follows. Complex carbohydrates are essentially made salt free, for example, by dialysis, gel filtration, or chromatography in a suitable system. Once unsalted, the HA is made essentially anhydrous, for example by lyophilization, and reduces moisture until equilibrium is achieved at least at 25 mbar lyophilization conditions at 25 ° C. The moisture of the hydrazine reagent is equally important and should not exceed 4% v / v. Thus, drying of the hydrazine reagent can be accomplished by one of a number of reported methods, but most commercial products of anhydrous hydrazine reagent are suitable. The appropriate hydrazine reagent is then added to the appropriately prepared sample in an airtight container. The reaction between HA and the hydrazine reagent can be initiated microscopically or macroscopically, for example, by energy input due to temperature rise. For any method of energy input, the optimum reaction conditions can be estimated from various experimental methods. In the present disclosure, the preferred method of initiating the reaction is by increasing the temperature to a steady state.

  In yet other embodiments, an alkaline solution can be used to remove N-linked glycans. The N-glycoside linkage that attaches the N-glycan to the conjugate is alkaline and unstable, and thus an alkaline solution can be used for N-glycan release. For example, as described in US Pat. No. 5,539,090, which is incorporated herein by reference in its entirety, the use of incubation with 1 M NaOH at 100 ° C. for 4 hours has been successful.

  It will be appreciated that a variety of influenza virus strains may be utilized as “sources” of genetic material encoding HA proteins suitable for use in the methods and compositions described herein. In various embodiments described herein, the modified HA proteins of the invention can be derived from any of the known HA sequences derived from influenza isolates known in the art. For example, the National Center for Biotechnology information (NCBI) maintains a database of known HA sequences (www.ncbi.nlm.nih.gov/gmomes/FLU/flu.html). In addition, a database for influenza HA wild type sequences or sequences passaged to MDCK cells and hen eggs is available from the Global Initiative on Sharing All Influenza Database (GISAID) EpiFlu database. Those skilled in the art can refer to these databases with reference to HA polypeptides in general and / or specific HA polypeptides (eg, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 polypeptides); or infection of specific hosts (eg, birds, camels, dogs, horses, cats, humans, leopards, minks, mice, seals, pigs, tigers, whales, etc.) Sequences unique to HA that mediate can be readily identified. Examples of subtypes comprising such HA proteins that can be modified by the methods described herein include, but are not limited to, A / New Caledonia / 20/99 (H1N1), A / Indonesia / 5/05 (H5N1 ), A / chicken / New York / 1995, A / herring gull / DE / 677/88 (H2N8), A / Texas / 32/2003, A / mallard / MN / 33/00, A / duck / Shanghai / 1 / 2000, A / northern pintail / TX / 828189/02, A / Turkey / Ontario / 6118/68 (H8N4), A / shoveler / Iran / G54 / 03, A / chicken / Germany / N / 1949 (H10N7), A / duck / England / 56 (H11N6), A / duck / Alberta / 60/76 (H12N5), A / Gull / Maryland / 704/77 (H13N6), A / Mallard / Gurjev / 263/82, A / duck / Australia / 341/83 (H15N8), A / black-headed gull / Sweden / 5/99 (H16N3), B / Lee / 40, C / Johannesburg / 66, A / PuertoRico / 8/34 (H1N1), A / Brisbane / 59/2007 (H1N1), A / Solomon Islands 3/2006 (H1N1), A / Brisbane 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2) , B / Malaysia / 2506/2004, B / Florida / 4/2006, A / Singapore / 1/57 (H2N2), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / HongKong / W312 / 97 (H6N1), A / Equine / Prague / 56 (H7N7), A / HongKong / 1073/99 (H9N2). A typical list of HA proteins that can be modified by the methods of the present invention can be found in US Patent Application Publication No. 2010/0239610, which is hereby incorporated by reference in its entirety. In one embodiment, the HA sequence selected for the modification of interest is an HA sequence that is compatible with chicken eggs. Methods for obtaining and identifying HA sequences compatible with hen's eggs are known in the art (Robertson et al, 1991, J. Gen. Virology 72: 2671-7).

  Furthermore, chimeric HA proteins comprising the transmembrane domain and / or cytoplasmic tail of a modified influenza HA protein fused to a protein from an infectious agent are included within the scope of the present invention. In another embodiment, the transmembrane domain and / or cytoplasmic tail of the modified influenza HA protein passes through the transmembrane domain to about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 to about 50 amino acids from the N-terminus or C-terminus and is fused to the protein from other infectious agents. Infectious agents may be viruses, bacteria, fungi, and / or parasites. Non-limiting examples of viruses from which the infectious agent protein can be derived include: coronavirus (eg, factors that cause SARS), hepatitis viruses A, B, C, D, and E3, human immunodeficiency virus (HIV), herpes virus 1, 2, 6, and 7, cytomegalovirus, shingles, papilloma virus, Epstein Barr virus, parainfluenza virus, respiratory syncytial virus (RSV), human metapneumo virus, adenovirus, Bunyavirus (eg hantavirus), coxsackie virus, picoma virus, rotavirus, rhinovirus, rubella virus, mumps virus, measles virus, rubella virus, poliovirus (s), adenovirus (s), Pareinph Enzavirus (multiple types), avian influenza (various types), shipping fever virus, Western and Eastern equine encephalomyelitis, Japanese encephalomyelitis, fowlpox, rabies virus, Slow brain virus, rous sarcoma virus, Papovaviridae, Parvoviridae, Picomaviridae, Poxviridae (such as pressure ulcers or vaccinia), reovirus Reoviridae (eg, rotavirus), Retroviridae (HTLV-I, HTLV-II, lentivirus), Togaviridae (eg, rubivirus), Newcastle disease virus, West Nile fever Virus, Tick borne encephalitis, yellow fever, Chikungunyau Irs and dengue viruses (all serotypes).

Vaccines containing modified HA proteins:
The application provides an influenza HA protein that has been modified to remove one or more glycosylation sites. In various aspects described herein, the modified HA proteins of the present invention have utility in immunogenic compositions such as vaccines for the treatment and / or prevention of influenza infection.

  Since influenza infection can be prevented by providing neutralizing antibodies to vertebrates, vaccines comprising modified influenza HA proteins can induce neutralizing antibodies in vivo when administered to vertebrates. The modified influenza HA protein is preferably used for the prevention and / or treatment of influenza infection. Accordingly, another aspect of this disclosure relates to a method for eliciting an immune response against influenza. The method includes administering to a subject (such as a human or animal subject) an immunologically effective amount of a composition containing a modified influenza HA protein. Administration of an immunologically effective amount of the composition elicits an immune response specific for the epitope present on the modified influenza HA protein. Such immune responses can include B cell responses (eg, production of neutralizing antibodies) and / or T cell responses (eg, production of cytokines). Preferably, the immune response elicited by the modified influenza HA protein comprises elements specific for at least one steric epitope present on the modified influenza HA protein.

  Vaccines comprising modified HA proteins within the scope of the present invention include inactivated influenza vaccines, live attenuated virus vaccines, and virus-like particles (VLPs).

  Three types of inactivated influenza vaccines are currently used worldwide: whole virus vaccines, split product vaccines, and surface antigen or “subunit” vaccines. All of these vaccines contain the surface glycoproteins of influenza virus strains, hemagglutinin (HA), and neuraminidase (NA) that are expected to spread into the human population in the coming season. These strains to be incorporated into the vaccine are grown in embryonated hen eggs and then the virus particles are purified for further processing. In one embodiment, to improve the immunogenicity of an existing influenza vaccine, one or more glycosylation sites are removed from HA sequences that are compatible with existing chicken eggs. Methods for preparing HA sequences that are compatible with chicken eggs are described in US Pat. No. 5,162,112, which is incorporated herein by reference in its entirety.

  Furthermore, the modified HA protein can be derived from one or more of influenza multivalent vaccines (eg, monovalent, divalent, or trivalent). In one aspect, the modified HA protein can be derived from one of the trivalent inactivated vaccines (TIV) against influenza. Standard components of TIV include hemagglutinin (HA) and neuraminidase from three different strains of influenza virus. Examples of TIV that can be used include, but are not limited to, Fluzone, Fluvirin, Fluarix, FluLaval, FluBlok, FluAd, Influvac, and Fluvax.

  In one aspect, the present invention provides a vaccine comprising a composition of modified HA proteins that is presented to the immune system and is capable of inducing an immune response in an individual. In some embodiments, the composition further comprises an adjuvant or carrier. In another embodiment, a modified HA protein described herein comprises one or more components of at least one trivalent inactivated influenza vaccine (TIV). In some embodiments, the TIV is selected from the group consisting of Fluzone, Fluvirin, Fluarix, FluLaval, FluBlok, FluAd, Influvac, and Fluvax. In further embodiments, the modified HA proteins described herein can be administered in conjunction with a TIV selected from the group consisting of Fluzone, Fluvirin, Fluarix, FluLaval, FluBlok, FluAd, Influvac, and Fluvax.

Virus-like particles containing a modified HA protein:
The present invention also provides an influenza virus-like particle (VLP) comprising a modified HA protein that can be formulated into a vaccine or antigen preparation to protect a vertebrate (eg, human) from influenza infection or at least one disease symptom thereof. ). VLPs include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, or fragments or portions thereof, one or two Modified HA proteins from one or more subtypes can be included. Examples of subtypes containing such HA proteins include A / New Caledonia / 20/99 (H1N1) A / Indonesia / 5/05 (H5N1), A / chicken / New York / 1995, A / herring gull / DE / 677/88 (H2N8), A / Texas / 32/2003, A / mallard / MN / 33/00, A / duck / Shanghai / 1/2000, A / northern pintail / TX / 828189/02, A / Turkey / Ontario / 6118/68 (H8N4), A / shoveler / Iran / G54 / 03, A / chicken / Germany / N / 1949 (H10N7), A / duck / England / 56 (H11N6), A / duck / Alberta / 60/76 (H12N5), A / Gull / Maryland / 704/77 (H13N6), A / Mallard / Gurjev / 263/82, A / duck / Australia / 341/83 (H15N8), A / black-headed gull / Sweden / 5/99 (H16N3), B / Lee / 40, C / Johannesburg / 66, A / PuertoRico / 8/34 (H1N1), A / Brisbane / 59/2007 (H1N1), A / Solomon Islands 3 / 2006 (H1N1), A / Brisbane 10/2007 (H3N2), A / Wisconsin / 67/2005 (H3N2), B / Malaysia / 2506/2004, B / Florida / 4/2006, A / Singapore / 1/57 ( H2N2 ), A / Anhui / 1/2005 (H5N1), A / Vietnam / 1194/2004 (H5N1), A / Teal / HongKong / W312 / 97 (H6N1), A / Equine / Prague / 56 (H7N7), A / HongKong / 1073/99 (H9N2).

  In aspects of the invention, the HA protein may be a H1, H2, H3, H5, H6, H7, or H9 subtype. In another aspect, the H1 protein is A / New Caledonia / 20/99 (H1N1) strain, A / PuertoRico / 8/34 (H1N1) strain, A / Brisbane / 59/2007 (H1N1) strain, or A / Solomon It may be derived from Islands 3/2006 (H1N1) strain. Further, the H3 protein may be derived from the A / Brisbane 10/2007 (H3N2) strain or the A / Wisconsin / 67/2005 (H3N2) strain. In a further aspect of the invention, the H2 protein may be derived from the A / Singapore / 1/57 (H2N2) strain. The H5 protein may be derived from the A / Anhui / 1/2005 (H5N1) strain, the A / Vietnam / 1194/2004 (H5N1) strain, or the A / Indonesia / 5/2005 strain. In an embodiment of the present invention, the H6 protein may be derived from A / Teal / HongKong / W312 / 97 (H6N1) strain. The H7 protein may be derived from the A / Equine / Prague / 56 (H7N7) strain. In an embodiment of the present invention, the H9 protein may be derived from the A / HongKong / l073 / 99 (H9N2) strain. In a further aspect of the invention, the HA protein may be derived from an influenza virus that may be a type B virus, including B / Malaysia / 2506/2004 or B / Florida / 4/2006.

  In some embodiments, the VLP comprising one or more modified influenza HA proteins further comprises additional influenza proteins such as M1, NA, M2, and NP. HA, NA, M1, M2, and NP proteins may be derived from any strain of influenza virus, including pandemic and seasonal strains.

  The VLPs of the invention can include avian influenza M1 protein. In one embodiment, the avian influenza M1 protein is derived from an H9N2 influenza strain. In various embodiments described herein, influenza M1 is A / quail / Hong Kong / Gl / 97, A / Hong Kong / 1073/99, A / Hong Kong / 2108/03, Duck / HK / Y280 / 97, CK / HK / G9 / 97, Gf / HK / SSP607 / 03, Ph / HK / CSW1323 / 03, WDk / ST / 4808/01, CK / HK / NT142 / 03, CK / HK / WF126 / 03 From, SCk / HK / WF285 / 03, CK / HK / YU463 / 03, CK / HK / YU577 / 03, SCk / HK / YU663 / 03, Ck / HK / CSWl61 / 03, and GF / HK / NTl01 / 03 It can be isolated from any one of the H9N2 influenza viruses selected from the group. In an exemplary embodiment, the H9N2 influenza strain is A / Hong Kong / 1073/99. In another embodiment, the avian influenza M1 protein is derived from an H5N1 influenza strain. In various embodiments described herein, influenza M1 is A / Vietnam / 1194/04, A / Vietnam / 1203/04, A / Hongkong / 213/03, A / Indonesia / 2/2005, A / It can be isolated from any one of the H5N1 influenza viruses selected from the group consisting of Bar headed goose / Quinghai / 1A / 2005, A / Anhui / 1/2005, and A / Indonesia / 5/05. In an exemplary embodiment, the H5N1 strain is A / Indonesia / 5/05.

  In another embodiment, a VLP of the invention can include influenza M1 protein from a non-avian source, such as from a Η1Η1 strain of porcine origin. In one embodiment, the M1 protein is derived from influenza A / California / 04/09. In other embodiments, the M1 protein may be derived from influenza A / California / 08/09 or influenza A / Mexico / 4108/09.

  The inventors have found that the use of an influenza M1 protein containing a YKKL L-domain sequence at the amino acid position corresponding to residues 100-103 of SEQ ID NO: 3 increases the efficiency of influenza VLP production. Thus, in an exemplary embodiment described herein, a VLP of the invention comprises an influenza M1 protein that contains a YKKL L-domain sequence. In one embodiment, the influenza M1 protein comprising a YKKL L domain sequence at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 3 is an avian influenza M1 protein. In another embodiment, the influenza M1 protein comprising a YKKL L domain sequence at the amino acid position corresponding to residues 100-103 of SEQ ID NO: 3 is a non-avian influenza M1 protein. In a further embodiment, the non-avian influenza M1 protein is a swine influenza M1 protein. In a further embodiment, the swine influenza M1 protein is derived from influenza strain A / California / 04/09. In an exemplary embodiment, the M1 protein from A / California / 04/09 consists of SEQ ID NO: 4. Methods for making VLPs that utilize M1 proteins carrying a putative YKKL L-domain are described in co-owned copending US patent application Ser. No. 12 / 832,657.

  Corresponding positions of the influenza M1 protein identified herein (eg, YKKL amino acids at positions corresponding to residues 100-103 of SEQ ID NOs: 3 and 4) are readily recognized by those of skill in the art for other influenza M1 proteins. Can be identified. Thus, given the designated regions and assays described in this application, one of skill in the art can identify native influenza M1 proteins that exhibit an increased ability to mediate VLP formation. Similarly, given the disclosure of this application, one of ordinary skill in the art will be able to make one or more modifications that increase the ability to mediate VLP formation in any influenza M1 protein of interest. Thus, a native influenza M1 protein having a YKKL amino acid at a position corresponding to residues 100-103 of SEQ ID NOs: 3 and 4 and a YKKL L-domain at a position corresponding to residues 100-103 of SEQ ID NOs: 3 and 4 Influenza M1 proteins that have been mutated, either naturally or by genetic engineering, are within the scope of the invention.

  In one embodiment, the VLP can include proteins from at least two different influenza viruses. For example, a VLP can include a modified HA (ie, Η5Ν1) from an avian influenza virus and NA from a swine influenza virus (ie, H1N1). In another embodiment, the VLP can comprise a modified HA protein derived from more than one type of influenza virus (ie, H5N1 and H1N1). In these embodiments, the VLP is a multivalent VLP capable of inducing an immune response against some proteins and thus against several strains of influenza virus. In another embodiment, the multivalent VLP comprises an influenza M1 protein. In one embodiment, the M1 protein comprises an L-domain sequence comprising YKKL at amino acid positions corresponding to residues 100-103 of SEQ ID NO: 3. In another embodiment, the M1 protein is swine influenza M1 protein. In a further embodiment, the swine influenza M1 protein is derived from influenza strain A / California / 04/09. In an exemplary embodiment, the M1 protein from A / California / 04/09 consists of SEQ ID NO: 4. In yet another embodiment, the M1 protein is an avian influenza M1 protein. In a further embodiment, the avian influenza M1 protein is derived from influenza strain A / Indonesia / 5/05. In an exemplary embodiment, the M1 protein from influenza strain A / Indonesia / 5/05 consists of SEQ ID NO: 3.

  The invention also encompasses variants of the protein expressed on or in the VLPs of the invention. Variants can contain changes in the amino acid sequence of the constituent proteins. The term “variant” with respect to a protein refers to an amino acid sequence in which one or more amino acids have changed relative to a reference sequence. Variants can have “conservative” changes in which the substituted amino acid has a similar structure or chemical property (eg, replacement of leucine with isoleucine). Alternatively, a variant can have “nonconservative” changes (eg, replacement of glycine with tryptophan). A minor number of similar variants can further include amino acid deletions or insertions, or both. Guidance on determining which amino acid residues can be substituted, inserted, or deleted without loss of biological or immunological activity is provided by computer programs well known in the art, such as DNASTA software. Can be found using.

  Natural variants can be caused by mutations in the protein. These mutations can lead to antigenic diversity within each group of infectious agents such as influenza. Thus, a person infected with an influenza strain will express an antibody against that virus when a new virus strain appears, and the antibody against the old strain will no longer recognize the new virus and reinfection may occur. The present invention encompasses all antigens and genetic diversity of proteins derived from infectious agents to make VLPs.

  General textbooks describing molecular biology techniques applicable to the present invention such as cloning, mutation, cell culture, etc. include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152 Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al, Molecular Cloning--A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2000 (“Sambrook” ), And Current Protocols in Molecular Biology, FM Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (“Ausubel”). These textbooks describe, for example, mutagenesis, vector usage, promoters, and many other related topics related to the cloning and mutation of HA, NA, and / or M1. Thus, the present invention also encompasses using known methods of protein engineering and recombinant DNA technology to improve or modify the properties of proteins expressed on or in the VLPs of the present invention. Various types of mutagenesis are used to generate and / or isolate mutant nucleic acids encoding protein molecules and / or to further modify / mutate proteins in or on the VLPs of the invention. be able to. These methods include, but are not limited to, site-specific, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil-containing templates, oligonucleotide directed mutagenesis, and phosphorothioate modification. Examples thereof include DNA mutagenesis and mutagenesis using double-stranded DNA with a gap. Further suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction selection and purification, deletion mutagenesis, mutagenesis by total gene synthesis, double-strand break repair, and the like. For example, mutagenesis involving a chimeric construct is also included in the present invention. In one embodiment, mutagenesis can be guided by known information about a natural molecule or a natural molecule that has been modified or mutated, such as sequence, sequence comparison, physical properties, crystal structure, and the like.

  The invention further includes protein variants that exhibit substantial biological activity, such as, when expressed on or in the VLPs of the invention, can elicit an effective antibody production response. Such variants include deletions, insertions, inversions, repeats and substitutions selected according to general rules known in the art so as to have little effect on activity.

  Methods for cloning the protein are known in the art. For example, a gene encoding a particular viral protein is isolated by RT-PCR of polyadenylated mRNA extracted from cells infected with the virus (DNA or RNA virus) or by PCR in cells infected with the DNA virus. be able to. The resulting product gene can be cloned into the vector as a DNA insert. The term “vector” refers to a means by which nucleic acids can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes and the like that can replicate autonomously or integrate into the host cell chromosome. Vectors are also not self-replicating, naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, polylysine-binding DNA or RNA, peptide-binding DNA or RNA Liposome-binding DNA or the like may be used. In many, but not all, general embodiments, the vectors of the invention are plasmids or bacmids.

  Accordingly, the present invention includes nucleotides encoding proteins cloned into expression vectors that can be expressed in cells that induce the formation of VLPs of the present invention. An “expression vector” is a vector such as a plasmid that can promote the expression and replication of a nucleic acid inserted therein. Typically, the expressed nucleic acid is “operably linked” to a promoter and / or enhancer and is subject to transcriptional control by the promoter and / or enhancer. In one embodiment, the vector comprises nucleotides encoding M1, NA, and modified HA.

  In some embodiments, the protein comprises a mutation that contains a modification that results in a silent substitution, addition, or deletion, but does not alter the properties and activities of the encoded protein nor the method of making the protein. Can do. Nucleotide variants may be generated for a variety of reasons, eg, to optimize codon expression for a particular host (changing the codons of human mRNA to those preferred for insect cells such as Sf9 cells). See US Patent Application Publication No. 2005/0118191, which is hereby incorporated by reference in its entirety for all purposes.

  In addition, nucleotides can be sequenced to clone the proper coding region to ensure that it does not contain any undesirable mutations. Nucleotides can be subcloned into an expression vector (eg, baculovirus) for expression in any cell. The above is just one example of how influenza proteins can be cloned. Those skilled in the art understand that still other methods are available and feasible.

  The present invention also provides a construct and / or vector comprising a nucleotide encoding the above influenza protein. Constructs and / or vectors containing influenza proteins include, but are not limited to, the AcMNPV polyhedrin promoter (or other baculovirus), lambda phage PL promoter, E. coli lac, phoA and tac promoters, SV40 early and It should be operably linked to an appropriate promoter, such as the late promoter, as well as the promoter of the retroviral LTR. 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 a transcription initiation site, termination site, and a ribosome binding site for translation in the transcribed region. The coding portion of the transcript expressed by the construct will preferably include a translation start codon at the start of the polypeptide to be translated and a stop codon appropriately located at the end.

  The expression vector will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin, or ampicillin resistance genes for culture in E. coli and other bacteria. Is mentioned. Among them, preferable vectors include baculovirus, pox virus (eg, vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoon pox virus, swinepox virus, etc.), adenovirus (eg, canine adenovirus), herpes There are viruses and viral vectors such as retroviruses. Other vectors that can be used in the present invention include those used in bacteria, which include pQE70, pQE60 and pQE-9, pBluescript vector, Phagescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223 -3, pKK233-3, pDR540, pRIT5. Among them, preferred eukaryotic vectors are pFastBac1 pWINEO, pSV2CAT, pOG44, pXT1 and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be readily apparent to those skilled in the art.

  The recombinant constructs described above can then be used for transfection, infection, or transformation and can express influenza proteins into eukaryotic and / or prokaryotic cells. Accordingly, the present invention provides a host cell comprising a vector containing a nucleic acid encoding an influenza protein, allowing expression of the construct in the host cell under conditions that allow the formation of VLPs.

  Among eukaryotic host cells are yeast, insects, birds, plants, C.I. There are 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 cells, such as High Five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells are S. cerevisiae. S. cerevisiae, Kluyveromyces lactis (K. lactis), C.I. C. albicans and C. albicans Candida species including C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris , As well as 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 oocytes or other cells of amphibian origin can also be used. Prokaryotic host cells include bacterial cells such as E. coli, B. pylori and the like. Examples include B. subtilis, and mycobacteria.

  A vector, eg, a vector comprising an influenza protein polynucleotide, can transfect a host cell according to methods well known in the art. For example, introduction of nucleic acids into eukaryotic cells can be accomplished by calcium phosphate coprecipitation, 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 nucleotides encoding one or more modified HA proteins and one or more additional influenza proteins selected from M1, NA, M2, and NP. . In an exemplary embodiment, the vector and / or host cell comprises one or more modified HA proteins and nucleotides encoding M1 and / or NA proteins. In another embodiment, the vector and / or host cell consists essentially of one or more modified HA proteins and M1 and / or NA proteins. In a further embodiment, the vector and / or host cell consists of one or more modified HA proteins and M1 and / or NA proteins. These vectors and / or host cells can contain additional markers, such as origins of replication, selectable markers.

  The present invention also provides constructs and methods that will further increase the efficiency of VLP production. For example, adding a leader sequence to the M1, HA, and / or NA protein can improve the efficiency of protein transport within the cell. For example, a heterologous signal sequence can be fused to M1, HA, and / or NA. In one embodiment, the signal sequence can be removed from the insect precursor protein gene and fused to M1, HA, and / or NA. In another embodiment, the signal peptide is a chitinase signal sequence, which functions efficiently in a baculovirus expression system.

  The invention also provides a method of producing a VLP of the invention, in a cell culture expression system, one or more additional HA proteins and one or more further selected from M1, NA, M2, and NP. A method comprising expressing a nucleic acid encoding an influenza protein and purifying said VLP from cell culture supernatant is provided.

  In some embodiments, one or more modified HA proteins and nucleic acids encoding M1 and / or NA proteins are expressed in eukaryotic cells under conditions that allow the formation of VLPs. In one embodiment, the eukaryotic cell utilized for expression is selected from the group consisting of yeast, insect, amphibian, avian, and mammalian cells. In an exemplary embodiment, the eukaryotic cell is an insect cell. In one embodiment, the cell culture expression system is a baculovirus expression system.

  Depending on the expression system and host cell selected, VLPs are produced by growing host cells transformed with the expression vector under conditions in which the recombinant protein is expressed and VLPs are formed. Selection of appropriate growth conditions is within the skill of the art or technician.

  Methods for growing cells engineered to produce the VLPs of the present invention include, but are not limited to, batch, batch-fed, continuous, and perfusion cell culture techniques. Cell culture means the growth and proliferation of cells in a bioreactor (fermentation chamber) in which the cells proliferate and express proteins for purification and isolation (eg, recombinant proteins). Typically, cell culture is performed in a bioreactor under aseptic, controlled temperature and atmospheric conditions. A bioreactor is a chamber used for cultured cells that can monitor environmental conditions such as temperature, air, agitation, and / or pH. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (eg, Cellbag®, Wave Biotech, Bridgewater, NJ). In another embodiment, the pre-sterilized plastic bag is between about 50L and 1000L.

  The VLP is then isolated using methods that retain its integrity, such as, for example, gradient centrifugation of cesium chloride, sucrose, iodixanol and standard purification techniques including, for example, ion exchange and gel filtration chromatography.

  The following are examples of methods for making, isolating and purifying the VLPs of the present invention. Usually, VLPs are produced from recombinant cell lines that have been engineered to form VLPs when the cells are grown in cell culture (see above). One skilled in the art will appreciate that there are additional methods that can be utilized to make and purify the VLPs of the invention, and thus the invention is not limited to the methods described.

Production of the VLPs of the present invention seeds Sf9 cells (uninfected) into shake flasks, allowing the cells to expand and scale up as cells grow and increase (eg, from a 125 ml flask to a 50 L wave bag ( Wave bag) can be started by). The medium used to grow the cells is that prepared for the appropriate cell line (preferably serum-free medium, eg insect medium ExCe11-420, JRH). The cells are then infected with recombinant baculovirus at the most efficient multiplicity of infection (eg, about 1 to about 3 plaque forming units per cell). Once infection occurs, M1, such as avian influenza M1, and at least one influenza heterologous protein (ie, the modified HA protein and / or NA protein of the present invention) are expressed from the viral genome, self-assembled into VLPs, and It is secreted from the cells after about 24-72 hours. In general, infection is most efficient when cells are in mid-log growth phase (4-8 × 10 ⁶ cells / ml) and at least about 90% are viable.

The VLPs of the present invention have approximately maximal VLP levels in the cell culture medium approximately 48-96 hours after infection, but can be recovered before extensive cell lysis occurs. The density and viability of Sf9 cells at the time of harvest is about 0.5 × 10 ⁶ cells / ml to about 1.5 × 10 ⁶ cells / ml, as indicated by dye exclusion assay, and at least 20% viability It can be. Next, the medium is removed and clarified. NaCl can be added to the medium to a concentration of about 0.4 to about 1.0M, preferably about 0.5M to avoid VLP aggregation. Removal of cells and cell debris from the cell culture medium containing the VLPs of the present invention is accomplished by tangential flow filtration (TFF) with a disposable pre-sterilized hollow fiber 0.5 or 1.00 μm filter cartridge or similar device. can do.

  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. Concentrated VLPs can be diafiltered against 10 volumes of phosphate buffered saline (PBS) at pH 7.0-8.0 containing 0.5 M NaCl to remove residual media components. it can.

  Concentrated and diafiltered VLPs are obtained at about 4 ° C. to about 10 ° C. on a 20% -60% discontinuous sucrose gradient in pH 7.2 PBS buffer containing 0.5 M NaCl. Further purification is possible by centrifuging at 500 × g for 18 hours. Typically, VLPs form distinct visible bands between about 30% to about 40% sucrose or at the interface (at 20% and 60% step gradients) that are recovered from the gradient and stored. can do. This product can be diluted to contain 200 mM NaCl in the preparation for the next step of the purification process. In addition to containing VLP, the product may contain intact baculovirus particles.

  Further purification of VLPs can be achieved by anion exchange chromatography or 44% isodensity sucrose cushion centrifugation. In anion exchange chromatography, a sample from a sucrose gradient (see above) is loaded onto a column packed with a medium containing an anion (eg, Matrix Fractogel EMD TMAE) and the VLP is loaded with other contaminants (eg, Eluting through a salt gradient (about 0.2 M to about 1.0 M NaCl), which can be separated from baculovirus and DNA / RNA). In the sucrose cushion method, a sample containing VLP is added to a 44% sucrose cushion and centrifuged at 30,000 g for about 18 hours. VLPs form a band at the top of 44% sucrose, while baculovirus precipitates at the bottom and other contaminating proteins remain in the top 0% sucrose layer. Collect VLP peaks or bands.

  If necessary, the intact baculovirus can be inactivated. Inactivation can be achieved by chemical methods such as formalin or β-propiolactone (BPL). Intact baculovirus removal and / or inactivation can be accomplished, for the most part, using selective precipitation and chromatographic methods known in the art, as exemplified above. it can. The inactivation method involves incubating a sample containing VLPs in 0.2% BPL for 3 hours at about 25 ° C. to about 27 ° C. Baculovirus can also be inactivated by incubating samples containing VLPs in 0.05% BPL at 4 ° C. for 3 days and then at 37 ° C. for 1 hour.

  After the inactivation / removal step, the product containing the VLP is passed through another diafiltration step to remove any reagents from the inactivation step and / or any residual sucrose, and the VLP is added to the desired buffer. (E.g., PBS). Solutions containing VLPs can be sterilized by methods known in the art (eg, filter sterilization) and stored in a refrigerator or freezer.

  The above technique can be implemented on a variety of scales. For example, T-type flasks, shake flasks, spinner bottles, and industrial scale bioreactors are possible. The bioreactor can include either a stainless steel tank or a pre-sterilized plastic bag (eg, a system sold by Wave Biotech, Bridgewater, NJ). Those skilled in the art will know what is most desirable for that purpose.

  Amplification and production of baculovirus expression vectors and production of recombinant influenza VLPs by recombinant baculovirus infection of cells can be accomplished in insect cells, eg, Sf9 insect cells, as described above. In a preferred embodiment, the cell is Sf9 infected with a recombinant baculovirus engineered to produce a VLP of the invention.

Pharmaceutical or vaccine formulation and administration:
In another aspect, the present invention provides an antigenic formulation comprising a VLP comprising one or more modified HA proteins. VLPs include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, or fragments or portions thereof, one or two Modified HA proteins from one or more subtypes can be included. In one embodiment, the VLP can include additional influenza virus proteins, including but not limited to M1, M2, NA, and NP. In an exemplary embodiment, the present invention includes an antigen formulation comprising a VLP comprising a modified HA protein, an M1 protein, and an NA protein. In one embodiment, a formulation of the invention is one or more described herein in combination with or formulated with one or more purified or partially purified modified HA antigens. VLPs can be included. For example, in one embodiment, the formulation comprises a VLP comprising a modified HA protein from influenza A / California / 04/09 in combination with a purified modified HA protein from the same virus. In another formulation, VLPs are combined with homologous or heterologous HA or NA. In another embodiment, separate formulations are made for at least one VLP and at least one HA or NA and administered simultaneously or separately to a patient or subject. In some embodiments, antibody production as measured by HA antibody titer or other scale is superior when VLP and HA are administered to the same patient or subject (or animal) in a single formulation or separate formulations. ing.

  The formulation of the present invention comprises the VLP of the present invention and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. A detailed discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (Mack Pub. Co. N.J. current edition). The formulation should be compatible with the method of administration. In another embodiment, the formulation is suitable for human administration and is preferably sterile, non-particulate, and / or non-pyrogenic.

  Pharmaceutical compositions useful herein include any pharmaceutical that does not itself induce the generation of an adverse immune response in a vertebrate administered with the composition, and can be administered without undue toxicity. Contains a pharmaceutically acceptable carrier including any suitable diluent or excipient, and a VLP of the invention. As used herein, the term “pharmaceutically acceptable” is either approved by federal or state government regulatory authorities, or, for use in mammals and more specifically in the United States Pharmacopeia, Means published in the European Pharmacopoeia or other generally accepted pharmacopoeias. These compositions can be useful as vaccines and / or antigen compositions for inducing a protective immune response in vertebrates.

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

  The present invention also includes a vaccine comprising a VLP comprising one or more modified HA proteins. VLPs include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, or fragments or portions thereof, one or two Modified HA proteins from one or more subtypes can be included. In one embodiment, the VLP can include additional influenza virus proteins, including but not limited to M1, M2, NA, and NP. In an exemplary embodiment, the invention includes a vaccine comprising a VLP comprising a modified HA protein, an M1 protein, and an NA protein.

  The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the vaccine formulation of the invention. In one embodiment, the kit includes two containers, one containing VLP and the other containing an adjuvant. In another embodiment, the kit includes two containers, one containing lyophilized VLP and the other containing a solution for resuspending the VLP. Such containers may be accompanied by notices in the form prescribed by government agencies that regulate the manufacture, use or sale of medicinal products or biological products, which notices are manufactured and used for human administration by the agency. , Or reflect sales approval.

  The present invention also provides that the VLP formulation is packaged in a sealed container such as an ampoule or sachet that indicates the amount of the composition. In one embodiment, the VLP composition is supplied to the subject as a liquid, and in another embodiment, as a dry sterilized lyophilized powder or a water-free concentrate, such as with water or saline to the subject. Can be reconstituted to a concentration suitable for administration. In one embodiment, the container comprises at least about 50 μg / ml, more preferably at least about 100 μg / ml, at least about 200 μg / ml, at least 500 μg / ml, or at least 1 mg / ml of a VLP associated antigen of the invention. .

  In another embodiment, the VLP composition is supplied in liquid form in a sealed container that indicates the quantity and concentration of the VLP composition. The liquid form of the VLP composition is sealed with at least about 50 μg / ml, more preferably at least about 100 μg / ml, at least about 200 μg / ml, at least 500 μg / ml, or at least 1 mg / ml of a VLP associated antigen of the invention. Supplied in a container.

  In general, the VLPs of the present invention are administered in an effective amount or an effective amount (as defined above) sufficient to stimulate an immune response against one or more infectious agents. Preferably, administration of a VLP of the invention elicits immunity against the infectious agent. Typically, dosages can be adjusted within this range based on, for example, age, health status, 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 needle-free injection device. Alternatively, the vaccine formulation is administered intranasally by either nasal fluid, large particle aerosol (greater than about 10 microns), or nebulization into the upper respiratory tract. Although any of the delivery routes described above generate an immune response, intranasal administration provides the added benefit of inducing mucosal immunity at the site of entry of many viruses, including influenza.

  Accordingly, the present invention also provides a method of formulating a vaccine or antigen composition that induces a mammal to immunize against an infection or at least one symptom thereof, wherein an effective dose of the VLP of the present invention is added to said formulation. Including methods.

  Methods for administering compositions comprising VLPs (vaccines and / or antigen formulations) include, but are not limited to, parenteral administration (eg, intradermal, intramuscular, intravenous, and subcutaneous), epidural anesthesia, mucosal (eg, Nasal and oral or pulmonary routes, or by suppositories). In certain embodiments, the compositions of the invention are administered intramuscularly, intravenously, subcutaneously, transdermally, or intradermally. The composition may be by any conventional route, such as by infusion or bolus injection, by epithelial or dermal mucosal lining (eg, oral mucosa, colon, conjunctiva, nasopharynx, oropharynx, sputum, urethra, bladder, and intestinal mucosa). Etc.) and may be administered with other biologically active agents. In some embodiments, intranasal or other mucosal routes of administration of a composition comprising a VLP of the invention can induce substantially higher antibodies or other immune responses than other routes of administration. In another embodiment, an intranasal or other mucosal route of administration of a composition comprising a VLP of the invention may induce an antibody or other immune response that induces cross protection against other strains or organisms that cause infection. it can. For example, VLPs containing influenza proteins can induce cross protection against several influenza strains when administered to vertebrates. Administration can be systemic or local.

  In yet another embodiment, the vaccine and / or antigen preparation is administered in such a way as to target mucosal tissue to elicit an immune response at the immune site. For example, mucosal tissues such as gut-associated lymphoid tissue (GALT) can be targeted for immunity by using oral administration of compositions containing adjuvants with specific mucosal targeting properties. Additional mucosal tissues such as nasopharyngeal lymphoid tissue (NALT) and bronchial associated lymphoid tissue (BALT) can also be targeted.

  The vaccines and / or antigen preparations of the present invention can also be administered, for example, on a schedule of initial administration of the vaccine composition followed by booster administration. In certain embodiments, the second dose of the composition is administered about 2 weeks to 1 year after the first dose, preferably about 1, about 2, about 3, about 4, about 5 months to about 6 months. The Further, the third dose is preferably about 4, about 5, or about 6 months or about 7 months after the second dose and after about 3 months to about 2 years or more after the first dose. Can be administered after about 1 year. The third dose is optionally administered after the second dose if no specific immunoglobulin is detected in the subject's serum and / or urine or mucus secretions or is detected at a low level. Can do. In a preferred embodiment, the second dose is administered about 1 month after the first dose, and the third dose is administered about 6 months after the first dose. In another embodiment, the second dose is administered about 6 months after the initial administration. In another embodiment, the VLPs of the invention can be administered as part of a combination therapy. For example, the VLPs of the present invention can be formulated with other immunogenic compositions, antiviral agents, and / or antibiotics.

  The dosage of the pharmaceutical formulation is, for example, a dose effective to elicit a prophylactic or therapeutic immune response, e.g. by measuring the serum titer of virus-specific immunoglobulin or by serum sample, urine sample, or One of skill in the art can readily determine by first identifying by measuring the inhibition rate of antibodies in mucosal secretions. The dosage can be determined from animal studies. A non-limiting list of animals used to test vaccine efficacy includes guinea pigs, hamsters, ferrets, chinchillas, mice, and cotton rats. Most animals are not natural hosts for infectious agents, but can still be useful for testing for various aspects of the disease. For example, in any of the above animals, a vaccine candidate, such as a VLP of the invention, may be dosed to partially characterize the immune response induced and / or which neutralizing antibodies are produced. Can be determined. For example, many tests are performed on mouse models because mice are small and their low cost allows researchers to conduct tests on a large scale.

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

Furthermore, as is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of non-specific stimulators of the immune response known as adjuvants. Adjuvants have been used experimentally to promote a general increase in immunity against unknown antigens (eg, US Pat. No. 4,877,611). For many years, immunization protocols have used adjuvants to stimulate responses, so adjuvants are well known to those skilled in the art. Some of the adjuvants affect the mode of antigen presentation. For example, precipitation of protein antigens with alum increases the immune response. Also, when the antigen is emulsified, the period of antigen presentation is extended. All its entirety by reference for purposes which are incorporated herein, Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2 nd Edition)" to be embraced any adjuvant described Are contemplated within the scope of the present invention.

  Typical adjuvants include complete Freund's adjuvant (a non-specific immune response stimulator containing dead Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant . Other adjuvants include GMCSP and BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Also contemplated is RIBI containing three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion. MF-59, Novasomes®, MHC antigens can also be used.

  In one embodiment of the present invention, the adjuvant is about 2 to 10 two, arranged in a substantially spherical shell form, separated by an aqueous layer surrounding a large amorphous central cavity that does not contain a lipid bilayer. It is a paucilamellar lipid vesicle with a stratified layer. Low-lamellar lipid vesicles can act to stimulate immune responses in several ways, as non-specific stimulators, as carriers for antigens, as carriers for additional adjuvants, and combinations thereof . For example, when a vaccine is prepared by mixing a pre-formed vesicle with an antigen such that the antigen remains extracellularly relative to the vesicle, the stratified membrane lipid vesicle acts as a nonspecific immunostimulator. By encapsulating the antigen in the central cavity of the vesicle, the vesicle acts as both an immunostimulant and an antigen carrier. In another embodiment, the vesicles are made primarily from non-phospholipid vesicles. In other embodiments, the vesicle is Novasomes®. Novasomes® are few multilamellar non-phospholipid vesicles ranging from about 100 nm to about 500 nm. This includes Brij 72, cholesterol, oleic acid, and squalene. Novasomes® has been shown to be an effective adjuvant against influenza antigens (US Pat. No. 5,629,021, incorporated herein by reference in its entirety for all purposes). No. 6,387,373 and 4,911,928).

  Another way of inducing an immune response can be achieved by formulating a VLP of the invention with an “immunostimulatory substance”. These are the body's own chemical mediators (cytokines) that increase the response of the immune system. Immunostimulants include, but are not limited to, interleukins (eg, IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (eg, granulocytes-macrophages ( GM) -colony stimulating factor (CSF)); and other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc. for immunostimulatory, immune enhancing and pro-inflammatory activities Examples include various cytokines, lymphokines, and chemokines. The immunostimulatory molecule may be administered in the same formulation as RSV VLP or may be administered separately. Either a protein or an expression vector encoding the protein can be administered to produce an immunostimulatory effect. Accordingly, in one embodiment, the present invention includes antigenic and vaccine formulations that include adjuvants and / or immunostimulants.

  Accordingly, one embodiment of the invention includes a formulation comprising a VLP and an adjuvant and / or immunostimulant. In another embodiment, the adjuvant is Novasomes®. In another embodiment, the formulation is suitable for human administration. In another embodiment, the formulation is administered to vertebrates orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously. In another embodiment, different VLPs are blended together to make a multivalent formulation. These VLPs may contain modified HA proteins from different influenza virus strains.

  Stimulation of immunity with a single dose is preferred, but additional dosages can be administered by the same or different routes to achieve the desired effect. In newborns and infants, for example, multiple doses may be required to elicit sufficient levels of immunity. Administration can be continued at certain intervals throughout childhood if necessary to maintain a sufficient level of protection from infection. Similarly, adults who are particularly susceptible to recurrent or severe infections, such as health care workers, day care workers, families of young children, the elderly, and individuals with impaired cardiopulmonary function, can establish protective immune responses and Multiple immunizations may be required for maintenance. The level of induced immunity can be monitored by measuring the amount of neutralizing secretions and serum antibodies, and dosages or vaccines can be prepared as necessary to induce or maintain the desired level of protection. The inoculation can be repeated.

Methods to stimulate an immune response:
As mentioned above, the VLPs of the present invention are useful in the preparation of compositions that stimulate an immune response that confers immunity to infectious agents. Both mucosal immunity and cellular immunity can contribute to immunity against infectious agents and diseases. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection. Secretory immunoglobulin A (sIgA) is involved in upper airway protection and serum IgG is involved in lower airway protection. The immune response induced by infection protects against reinfection with the same virus or antigenically similar virus strain. For example, influenza undergoes frequent and unpredictable changes; therefore, after natural infection, the effective period of protection provided by host immunity may be only a few years against new strains of viruses that spread in the region .

  When administered to a vertebrate (eg, a human), the VLP of the present invention can induce immunity in the vertebrate. Immunity arises from an immune response to the VLPs of the invention that protects or ameliorates the infection or at least reduces the symptoms of the infection in the vertebrate. In some cases, when the vertebrate is infected, the infection becomes asymptomatic. The response may not be a full defense response. In this case, when the vertebrate is infected with an infectious agent, the vertebrate will experience reduced symptoms or a shorter symptom period compared to a non-immune vertebrate.

  The present invention includes a method of inducing immunity in a vertebrate comprising administering to said vertebrate a VLP comprising one or more modified HA proteins. VLPs include H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16, or fragments or portions thereof, one or two Modified HA proteins from one or more subtypes can be included. In one embodiment, the VLP can include additional influenza virus proteins, including but not limited to M1, M2, NA, and NP. In an exemplary embodiment, the invention includes a vaccine comprising a VLP comprising a modified HA protein, an M1 protein, and an NA protein. In one embodiment, the immune response induced is a humoral immune response. In another embodiment, the immune response induced is a cellular immune response.

  As used herein, an “antibody” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical polypeptide chain pairs, each pair having one “light” chain (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that plays a major role in antigen recognition. Antibodies exist as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases.

  In another embodiment, the invention relates to a method of inducing a protective cell response against infection or at least one symptom thereof in a subject, wherein said VLP of the invention comprises one or more modified HA proteins. Including a method comprising administering at least one effective dose. VLPs include H1, H2, H3, H4, H5, H6, H7, H8, H9, H1O, H11, H12, H13, H14, H15, or H16, or fragments or portions thereof, one or two Modified HA proteins from one or more subtypes can be included. In one embodiment, the VLP can include additional influenza virus proteins, including but not limited to M1, M2, NA, and NP. In an exemplary embodiment, the invention includes a vaccine comprising a VLP comprising a modified HA protein, an M1 protein, and an NA protein.

  As described above, the VLPs of the present invention can prevent or reduce at least one symptom of influenza in a subject when administered to the subject. Most symptoms of influenza are well known in the art. Accordingly, the methods of the present invention include the prevention or alleviation of at least one symptom associated with influenza. Symptom relief can be determined subjectively or objectively, eg, by self-assessment by a subject, by clinician assessment, or by performing an appropriate assay or measurement (eg, body temperature). For example, assessment of quality of life, delay of progression of infection or further symptoms, reduction of severity of symptoms, or appropriate assays (eg, antibody titers and / or T cell activation assays). Objective assessment includes both animal and human assessment.

  The present invention includes a method for preventing and / or reducing infection by influenza or symptoms thereof, comprising administering the VLP of the present invention to the vertebrate.

  A strategy for controlling infection during an outbreak (eg, influenza) is to vaccinate healthy individuals, including children, uniformly. For example, vaccination of circulating influenza vaccines to approximately 80% of local students reduced respiratory disease in adults and excess mortality in the elderly (Reichert et al., 2001). This concept is known as community immunity or “collective immunity” and is thought to play an important role in protecting the community from disease. Because a person who has been vaccinated has an infectious agent, such as an antibody that neutralizes influenza virus, the person is very unlikely to transmit the factor to another person. Thus, people who have not been vaccinated (and those who are less effective or who are not fully effective) are not sick of the people who have received the vaccine around them So it can often be protected by mass immunity. The higher the proportion of people who are vaccinated, the more effective immunity is. It is believed that about 95% of people in the region must be protected by vaccines to achieve mass immunity. Unimmunized people increase their chances of getting the disease and others.

  Thus, the present invention is also a method of reducing the severity of influenza in a population, wherein the VLPs of the present invention may be administered to sufficient individuals in the population and transmitted to other individuals in the population. Including a method comprising preventing or reducing. The present invention also provides for a population or region by administering a VLP of the present invention to a local population in order to reduce the incidence of infection between immunocompromised individuals or individuals who have not been vaccinated. A method of inducing immunity against influenza virus is included. In one embodiment, most school-age children are immunized by administration of the VLPs of the invention. In another embodiment, administration of the VLPs of the present invention immunizes most healthy individuals in the area. In another embodiment, the VLPs of the present invention are part of a “dynamic vaccination” strategy. Dynamic vaccination is the stable generation of low potency vaccines that are related to emerging pandemic strains but cannot provide complete protection to mammals due to antigenic variation (Germann et al., 2006). checking).

  The invention is illustrated in more detail by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as figures and sequence listings, are incorporated herein by reference for all purposes.

Example 1
Generation of Influenza Virus-Like Particles (VLPs) Containing Modified Hemagglutinin (HA) Protein This example produces influenza H5N1 VLPs that are made with HA proteins that have been modified to remove one or more glycosylation sites Is described. To construct a modified HA protein, one or both of the NX (T / S) sequences at amino acid positions 170-172 and 181-183 of the A / Indonesia / 05/2005 (H5N1) HA1 (SEQ ID NO: 2) protein Was mutated (FIG. 1). Specifically, a mutation from threonine to alanine was made at positions 172 and / or 183 of the HA1 protein.

  After modifying the HA gene, the modified HA gene was co-expressed with NA and M1 genes from A / Indonesia / 5/2005 (H5N1). The HA, NA, and M1 genes were expressed in Spodoptera frugiperda Sf9 insect cells using a baculovirus expression system. The expression product of infected Sf9 cells was characterized by SDS-PAGE analysis. As shown in FIG. 2, predicted molecular weight HA, NA, and M1 proteins (64 kd, 60 kd, and 31 kd, respectively) were detected. The presence of M1 and HA bands in the same lane indicates HA associated with M1 aggregates that are evidence of VLP formation. Modified to remove glycosylation sites at amino acid positions 170-172 (lanes 4-6), amino acid positions 181-183 (lane 2), or both amino acid positions 170-172 and 181-183 (lane 3) VLPs containing HA protein formed normally. These data provide evidence that it is possible to create influenza VLPs containing HA modified to remove one or more glycosylation sites, which alters the glycosylation pattern This is a surprising result considering the reports of changes in protein conformation that follow.

Example 2
Influenza VLPs containing modified hemagglutinin (HA) protein retain hemagglutinin and neuraminidase activity In this example, the hemagglutinin and neuraminidase activities of HA and NA proteins in wild type A / Indonesia / 05/2005 (H5N1) VLPs, respectively, A / Indonesia / 05/2005 from an H5N1 VLP containing an HA protein modified to remove one or more glycosylation sites at positions 170-172 and 181-183 of the HA protein (SEQ ID NO: 2) Compared to the same activity.

  To measure the hemagglutination activity of influenza VLPs, a 2-fold dilution series of sucrose gradient fractions containing wild type influenza VLPs or modified HA protein-containing influenza VLPs was prepared. These VLPs were then mixed with guinea pig erythrocytes in PBS and incubated at 4 ° C. for 1-16 hours. The degree of hemagglutination was visually determined to determine the highest dilution that could agglutinate guinea pig erythrocytes. The highest hemagglutination titer observed for wild type influenza VLPs was 1: 4096. In comparison, influenza VLPs containing a single T172A glycosylation site-modified HA protein exhibited a hemagglutination titer of 1: 2048, while influenza VLPs containing T172A and T183A glycosylation site-modified HA proteins , Showed a hemagglutination value of 1: 1024. These results demonstrate that H5N1 VLPs containing HA proteins modified to remove one or more glycosylation sites retain hemagglutination activity.

  In addition, the amount of neuraminidase activity in the influenza VLP-containing sucrose gradient fraction was measured using a neuraminidase assay in which NA protein acts on the substrate fetuin to release sialic acid. Using thiobarbituric acid that produces a pink color in proportion to the amount of free sialic acid, the amount of released sialic acid is chemically determined, and the amount of chromophore is measured using a spectrophotometer at a wavelength of 594 nm To do. Neuraminidase activity is detected in wild-type influenza VLPs, VLPs containing a single T172A glycosylation site-modified HA protein, and influenza VLPs containing T172A and T183A glycosylation site-modified HA proteins. It has been shown that H5N1 VLPs containing HA proteins modified to remove chain addition sites retain hemagglutination activity.

Example 3
Identification of N-linked glycosylation sites using NetNGlyc 1.0 software In this example, NetNGlyc predictive analysis software was used to identify N-linked glycosylation sites at the top of the globular head in three seasonal influenza strains. Identified.

  The first analytical strain in this example was A / California / 07/2009 (H1N1). NetNGlyc prediction software identified glycosylation sites at position 28 (along with the accompanying amino acid sequence “NSTD”), position 40 (NVTV), position 304 (NTSL), and position 557 (NGSL). None of these sites are located in the “top” region. Therefore, HA derived from pandemic A / California / 07/2009 (H1N1) does not have a glycosylation site in the region near the top of the HA spherical head.

  The second analytical strain in this example was A / Perth / 16/2009 (H3N2). NetNGlyc prediction software is ranked 24th (NSTA), 38th (NGTI), 79th (NCTL), 142th (NWTG), 149th (NGTS), 181st (NVTM), 262th (NSTG), and 301st The glycosylation site was identified in (NGSI). Of these sites, the following 4 sites are located in the “parietal” region: 142, 149, 181, and 262. Based on these results, the following four proposed mutation sites were suggested: N142D, T151A, T183A, and N262D. Of these four proposed mutations, N142D, T151A, and N262D are naturally occurring. In contrast, the T183A mutation does not exist in nature, suggesting that this glycosylation site is extremely conserved.

  The third analytical strain in this example was B / Brisbane / 60/2008. NetNGlyc prediction software is ranked 40th (NVTG), 74th (NCTD), 160th (NITN), 181st (NKTA), 212th (NETQ), 248th (NQTE), 319th (NKSK), 348th ( NGTK) and glycosylation sites at position 578 (NVSC) were identified. Of these sites, the following 4 sites are located in the “parietal” region: 160, 181, 212, and 248. Based on these results, the following four proposed mutation sites were suggested: T162A, T183A, T214A (chicken egg-based mutation), and N248D. Of these four proposed mutations, T214A and N248D are naturally occurring. In contrast, the T162A and T183A mutations do not exist in nature, suggesting that this glycosylation site is highly conserved.

  The foregoing detailed description has been presented for clarity of understanding only, and modifications should be apparent to those skilled in the art and should not be construed as having unnecessary limitations therefrom. None of the information provided herein is admitted to be prior art or related art to the claimed invention, and any publication specifically or implicitly referenced is prior art. It does not admit that it is.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

  Although this application is divided into sections to direct the reader's attention to specific embodiments, such sections should not be construed as a division between the embodiments. The teachings of each section and the embodiments described therein are applicable to other sections.

  Although the present invention has been described with reference to specific embodiments thereof, further modifications are possible, and the present application generally follows the principles of the present invention and is known or within the scope of the technical field to which the present invention pertains. Any variation of the present invention, including departures from the present disclosure, as included in the usual embodiments, and to which the essential features shown above and according to the appended claims may be applied It will be understood that it is intended to cover forms, uses, or adaptations.

Claims (46)

  A virus-like particle (VLP) comprising a recombinant influenza virus hemagglutinin (HA) protein, wherein the HA protein has one or more amino acid mutations that remove one or more glycosylation sites from the HA protein. VLP containing.   The VLP of claim 1, wherein the HA protein is derived from an avian influenza virus strain.   The VLP of claim 2, wherein the avian influenza virus strain is H5N1.   The VLP of claim 2, wherein the avian influenza virus strain is H9N2.   The VLP of claim 1, wherein the HA protein is derived from a swine influenza virus strain.   6. The VLP of claim 5, wherein the swine influenza virus strain is H1N1.   2. The VLP of claim 1, wherein the HA protein is derived from a seasonal influenza virus strain.   The VLP of claim 7, wherein the seasonal influenza virus strain is an influenza A virus strain.   8. The VLP of claim 7, wherein the seasonal influenza virus strain is a type B influenza virus strain.   The VLP of claim 1, wherein the HA protein is derived from a pandemic influenza virus strain.   The VLP of claim 1, wherein the HA protein is derived from a prepademic influenza virus strain.   The VLP according to any one of claims 1 to 11, wherein the HA protein exhibits hemagglutinin activity.   The VLP according to any one of claims 1 to 12, wherein the HA protein is a chimeric protein.   14. A VLP according to any one of the preceding claims, wherein the one or more amino acid mutations occur in the receptor binding domain (RBD) of the HA protein.   The one or more amino acid mutations change the NX (T / S) (SEQ ID NO: 1) sequence in the HA protein, X is any amino acid, and T / S is a threonine or serine residue. The VLP according to any one of claims 1 to 14, which is a group.   16. The VLP of claim 15, wherein the amino acid mutation is located at an amino acid position corresponding to an amino acid residue selected from 170-172 and 181-183 of SEQ ID NO: 2.   16. A VLP according to any one of the preceding claims, wherein the VLP comprises an influenza matrix (M1) protein.   18. A VLP according to claim 17, wherein the M1 protein is derived from an avian influenza virus strain.   19. A VLP according to claim 18, wherein the avian influenza virus strain is H9N2.   19. The VLP of claim 18, wherein the avian influenza virus strain is H5N1.   21. The VLP of claim 20, wherein the avian influenza virus strain is A / Indonesia / 5/05.   The VLP of claim 21, wherein the M1 protein from A / Indonesia / 5/05 comprises SEQ ID NO: 3.   18. A VLP according to claim 17, wherein the M1 protein is derived from a swine influenza virus strain.   24. The VLP of claim 23, wherein the swine influenza virus strain is H1N1.   18. The VLP of claim 17, wherein the M1 protein comprises an amino acid residue YKKL at amino acid positions corresponding to positions 100 to 103 of SEQ ID NO: 3.   26. A VLP according to any one of the preceding claims, wherein the VLP comprises an influenza neuraminidase (NA) protein.   27. The VLP of claim 26, wherein the NA protein exhibits neuraminidase activity.   28. Any of claims 1-27, wherein the VLP induces a stronger immune response in a mammal than a parent VLP comprising an HA protein that is not mutated to remove one or more glycosylation sites. The VLP according to one item.   A vaccine comprising the VLP according to any one of claims 1 to 28, wherein the vaccine induces substantial immunity against influenza virus infection in a mammal susceptible to influenza infection.   30. The vaccine of claim 29, wherein the mammal is a human.   A method of inducing substantial immunity against influenza virus infection in a mammal susceptible to influenza infection, comprising at least one effective dose of a vaccine comprising the VLP according to any one of claims 1 to 28. Administering to a mammal.   32. The method of claim 31, wherein the mammal is a human.   A method of formulating a vaccine that induces substantial immunity against influenza virus infection in a mammal susceptible to influenza infection, wherein an effective dose of VLP according to any one of claims 1 to 28 is added to the formulation. A method wherein the vaccine induces substantial immunity against influenza virus infection in the mammal. A method of making a VLP, comprising:
a) constructing a recombinant baculovirus construct encoding a recombinant influenza virus hemagglutinin (HA) protein, wherein the HA protein removes one or more glycosylation sites from the HA protein Or a process comprising a plurality of amino acid mutations;
b) transfecting, infecting or transforming a suitable host cell with the recombinant baculovirus and culturing the host cell under conditions that allow expression of the HA protein;
c) allowing the formation of VLPs in said host cell;
d) recovering the infected cell medium containing the VLP;
e) purifying the VLP.   35. The method of claim 34, wherein the HA protein is derived from an avian influenza virus.   36. The method of claim 35, wherein the avian influenza virus is H5N1.   36. The method of claim 35, wherein the avian influenza virus is H9N2.   35. The method of claim 34, wherein the HA protein is derived from swine influenza virus.   40. The method of claim 38, wherein the swine influenza virus is H1N1.   35. The method of claim 34, wherein the HA protein is derived from a seasonal influenza virus.   41. The method of claim 40, wherein the seasonal influenza virus is an influenza A virus.   41. The method of claim 40, wherein the seasonal influenza virus is a type B influenza virus.   43. The method according to any one of claims 34 to 42, wherein the HA protein is a chimeric protein.   44. The method of any one of claims 34 to 43, wherein the one or more amino acid mutations occur within a receptor binding domain (RBD) of the HA protein.   The amino acid mutation changes the NX (T / S) (SEQ ID NO: 1) sequence in the HA protein, X is any amino acid, and T / S is a threonine or serine residue. Item 45. The method according to any one of Items 34 to 44.   46. The method of claim 45, wherein the amino acid mutation is located at an amino acid position corresponding to an amino acid residue selected from 170-172 and 181-183 of SEQ ID NO: 2.

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