Broadly protective inactivated influenza virus vaccine

Abstract

A universal influenza virus vaccine composition comprising a plurality of inactivated influenza A viruses is described. The composition comprises four or more different influenza A viruses, each with a different hemagglutinin (HA) subtype. The vaccine composition can be used, for example, to elicit an immune response to influenza viruses, to immunize a subject against seasonal influenza viruses, and / or to mitigate future pandemics by serving as a pre-pandemic vaccine.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 620,051, filed January 22, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to compositions comprising a mixture of inactivated, low-pathogenic influenza virus strains and their use to elicit a broadly reactive immune response against seasonal influenza and to suppress zoonotic influenza pandemics. Background Art

[0004] Influenza viruses are members of the Orthomyxoviridae family. There are three types of influenza viruses, designated influenza A, B, and C. Influenza A viruses infect not only humans but also many birds and mammals and are classified by the subtypes of their surface proteins, hemagglutinin (HA) and neuraminidase (NA). Influenza A virions contain a segmented, negative-sense RNA genome that encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1), proton ion channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB1), PB1-F2, polymerase basic protein 2 (PB2), polymerase acidic protein (PA), PA-X, nonstructural protein 1 (NS1), and nonstructural protein 2 (NS2). HA, NA, M1, and M2 proteins are membrane-associated proteins, while NP, PB1, PB2, and PA are nucleocapsid-associated proteins, and PB1-F2, NS2, and PA-X proteins are nonstructural proteins. HA and NA proteins are envelope glycoproteins and are the source of the main immunodominant epitopes for virus neutralization and protective immunity, where HA is responsible for virus attachment and penetration of virus particles into cells, and NA is responsible for virus release.

[0005] The public health burden of influenza is substantial, with an average of over 200,000 hospitalizations and deaths from seasonal influenza in the United States ranging from 3,000 to 80,000 annually. These totals can increase significantly during pandemic years. In 1918, the worst influenza pandemic in history, 675,000 people died in the United States and as many as 50 to 100 million worldwide. Furthermore, novel influenza HA and NA subtypes (to which most people have no immunity) can emerge in animals (such as poultry and pigs) and spread to humans. These can cause zoonotic outbreaks, leading to pandemics. For example, a human-adapted swine H1N1 virus caused the 2009 pandemic. Avian-adapted strains H5N1, H9N2, H7N9, H10N8, and H6N1 have all caused human infections, often with significant mortality rates.

[0006] Since the 2009 pandemic, zoonotic infections with H5N1, H7N9, H3N2, and more recently H6N1 and H10N8 have been observed, increasing the need for a broadly reactive or universal vaccine approach that extends beyond protection against defined circulating seasonal variants to help prevent or mitigate future pandemics as a pre-pandemic vaccine. Live attenuated vaccines are problematic because they are overly attenuated and have strict guidelines for use. In addition, live viruses expressing hemagglutinin (HA) and / or neuraminidase (NA) subtypes that are not present in seasonal strains cannot be used because of the risk of reassortment with wild-type viruses. Therefore, a broadly reactive vaccine that can generate a protective immune response without the need for the use of live attenuated viruses is needed. The major difficulty faced by universal influenza vaccine approaches is the antigenic variability of different HA and NA subtypes. Universal vaccines can serve as pre-pandemic vaccines, providing protection against zoonotic influenza infections, and providing protection against seasonal influenza strains, or both. Summary of the Invention

[0007] Described herein is a universal influenza virus vaccine composition comprising a mixture of low pathogenicity, inactivated influenza viruses. The composition comprises four or more different influenza A viruses, each with a different hemagglutinin (HA) subtype, and each virus is monovalent (e.g., each virus has only one HA subtype). The vaccine composition can be used, for example, to elicit an immune response to influenza viruses or to immunize a subject against seasonal influenza viruses. The disclosed influenza virus composition can also be used as a pre-pandemic vaccine to prevent or mitigate future zoonotic influenza pandemics.

[0008] A composition is provided comprising: a first influenza A virus having a first subtype HA; a second influenza A virus having a second subtype HA; a third influenza A virus having a third subtype HA; and a fourth influenza A virus having a fourth subtype HA, wherein the four different influenza A viruses are inactivated. In some embodiments, the composition further comprises a fifth, sixth, seventh, and / or eighth inactivated influenza A virus, each virus having a different HA subtype. In some instances, the first, second, third, fourth, fifth, sixth, seventh, and / or eighth influenza viruses have HA subtypes selected from any of the following: H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. In some instances, the viruses are chemically inactivated. Each virus in the composition has only a single HA subtype (e.g., is monovalent).

[0009] Also provided are containers, such as syringes or vials, comprising the compositions disclosed herein. Also provided are kits comprising the disclosed containers.

[0010] A method for inducing an immune response against influenza virus in a subject by administering a therapeutically effective amount of the disclosed composition is further provided. A method for immunizing a subject against influenza virus by administering a therapeutically effective amount of the disclosed composition is also provided. In some instances, the obtained immune response is heterosubtypic, i.e., immune protection against influenza subtypes not present in the composition administered to the subject is achieved (e.g., administration of a composition carrying influenza viruses of subtypes H1, H3, H5, and H7HA (which may be chemically inactivated), and achieving an immune response or protection against influenza subtypes H2, H4, H6, and / or H10).

[0011] The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure shows the reciprocal hemagglutination inhibition (HAI) titers in mice vaccinated with PBS (negative control), a quadrivalent influenza virus-like particle (VLP) vaccine, or a β-propiolactone (BPL)-inactivated whole-virus quadrivalent influenza vaccine. Titers against H1N1, H3N8, H5N1, and H7N3 are shown. Both vaccines include HA from H1, H3, H5, and H7 influenza.

[0013] Figure 2AThis is a schematic diagram showing the timeline for vaccination of mice with a mixture of BPL-inactivated, low-pathogenic avian influenza viruses. Mice were inoculated intranasally (IN) or intramuscularly (IM) with a mixture of four different BPL-inactivated influenza viruses, each expressing a different avian HA (H1, H3, H5, or H7). Mice received a booster immunization 3 weeks (21 days) after primary immunization and were challenged 6 weeks (50 days) with multiple different subtypes of influenza virus.

[0014] Figure 2B is a series of graphs showing the survival of vaccinated mice challenged with H6N1, H7N1, H10N1 or H10N7 influenza viruses. All mice vaccinated with either IN or IM vaccine survived each influenza virus challenge, while mock-vaccinated mice died 6-8 days after challenge.

[0015] Figure 3 Dendrogram including two hemagglutinin (HA) subtypes showing the composition of quadrivalent vaccine 1 (containing HA subtypes H1, H3, H5 and H7) and quadrivalent vaccine 2 (containing HA subtypes H2, H4, H9 and H10).

[0016] Figures 4A-4E The results were as follows: blank vaccination, IN vaccination with quadrivalent (H2 / H4 / H9 / H10) BPL-inactivated influenza virus vaccine, or IM vaccination with heterotypic virus H1N1 ( Figure 4A ), H6N1( Figure 4B ), H7N1( Figure 4C ) and H15N1( Figure 4D ) or the same virus H10N1 ( Figure 4E ) A series of graphs showing the survival rate of mice challenged with .

[0017] Figure 5A are a pair of graphs showing nasal titers five days after challenge with pathogenic strains A / Port Chalmers / 1971 (H3N2) (left) or A / swine / Iowa / 1931 (H1N1) (right) in ferrets vaccinated IN or IM with a BPL-inactivated quadrivalent (H1 / H3 / H5 / H7) influenza virus vaccine.

[0018] Figure 5B Shown as Figure 5A Lung pathology in ferrets vaccinated and challenged as described. DETAILED DESCRIPTION

[0019] I. Abbreviations

[0020] BPL β-propiolactone

[0021] HA hemagglutinin

[0022] HAI Hemagglutination Inhibition

[0023] HAU hemagglutination unit

[0024] IAV influenza A virus

[0025] IBV influenza B virus

[0026] ICV influenza C virus

[0027] IM intramuscular

[0028] IN intranasal

[0029] NA neuraminidase

[0030] NI Neuraminidase Inhibitor

[0031] NP nuclear protein

[0032] NS Non-structural

[0033] RBC Red blood cells

[0034] RDE receptor destroying enzyme

[0035] RT Room temperature

[0036] VLP virus-like particles

[0037] WHO World Health Organization

[0038] II. Terminology and Methodology

[0039] Unless otherwise specified, technical terms are used according to their conventional usage. Definitions of commonly used terms in molecular biology can be found in the following references: Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0040] To facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0041] Adjuvant: a substance or vehicle that non-specifically enhances the immune response to an antigen (e.g., an influenza virus antigen). An adjuvant can be used with a composition disclosed herein, for example, a portion of a pharmaceutical influenza virus vaccine composition provided herein. An adjuvant may include a suspension of a mineral (alum, aluminum hydroxide, or phosphate) onto which an antigen is adsorbed; or an oil-in-water emulsion (e.g., Freund's incomplete adjuvant) in which an antigen solution is emulsified in mineral oil, sometimes including killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those comprising a CpG motif) can also be used as adjuvants (e.g., see U.S. Patents 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705 and 6,429,199). Adjuvants also include biomolecules, such as costimulatory molecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41BBL. In one example, the adjuvant is one or more toll-like receptor (TLR) agonists, such as agonists of TLR1 / 2 (which can be synthetic ligands) (e.g., Pam3Cys), TLR2 (e.g., CFA, Pam2Cys), TLR3 (e.g., polyI:C, poly A:U), TLR4 (e.g., MPLA, lipid A and LPS), TLR5 (e.g., flagellin), TLR7 (e.g., gardiquimod, imiquimod, loxoribine, ), TLR7 / 8 (e.g., R0848), TLR8 (e.g., imidazoquinoline, ssPolyU, 3M-012), TLR9 (e.g., ODN 1826 (Type B), ODN 2216 (Type A), CpG oligonucleotides), and / or TLR11 / 12 (e.g., profilin). In one example, the adjuvant is lipid A, such as monophosphoryl lipid A (MPL) from Salmonella serotype Minnesota Re 595 (e.g., Sigma Aldrich Catalog # L6895).

[0042] Administration: As used herein, administering a composition (such as those comprising an influenza virus composition) to a subject means giving the composition to, applying to, or exposing the composition to the subject. Administration can be achieved through any of a variety of routes, such as, for example, intramuscular, intranasal, pulmonary, local, oral, subcutaneous, intraperitoneal, intravenous, intrathecal, rectal, vaginal, and intradermal.

[0043] Antibody: An immunoglobulin molecule with a specific amino acid sequence produced by B lymphoid cells. Antibodies are induced in humans or other animals by specific antigens (immunogens, such as influenza virus HA and NA). Antibodies are characterized by their specific reaction with the antigen in a demonstrable manner, with the antibody and antigen being defined relative to each other. "Eliciting an antibody response" refers to the ability of an antigen or other molecule to induce antibody production.

[0044] Antigens or immunogens: compounds, compositions, or substances that can stimulate the production of antibody or T-cell responses in an animal, including compositions injected or absorbed into the animal. Antigens are products of specific humoral or cellular immunity, including those induced by heterologous immunogens.

[0045] β-Propiolactone (BPL): An organic compound from the lactone family with a four-membered ring. BPL is a chemical inactivator of infectious agents, including viruses, used in vaccines. This chemical is an alkylating agent that reacts with a wide range of nucleophiles, including nucleic acids and proteins. BPL treatment can induce nicks in DNA and crosslinks between nucleic acids and proteins. BPL inhibits influenza virus fusion (Bonnafous et al., Biochim Biophys Acta 1838(1Pt B):355-363, 2013). The chemical formula of BPL is C3H4O2. BPL is also known as oxetane-2-one, 3-hydroxypropiolactone, propiolactone, β-propiolactone, and 2-oxetanone.

[0046] Hemagglutinin (HA): A glycoprotein on the surface of the influenza virus. HA mediates the binding of viral particles to host cells and subsequent viral entry into host cells. HA also induces erythrocyte agglutination. HA (along with NA) is one of the two main antigenic determinants of influenza viruses.

[0047] Heterosubtypic immune response: An immune response elicited by infection or immunization with influenza A virus that provides protection against infection with an influenza virus having another HA or NA subtype.

[0048] Immune response: The reaction of cells of the immune system, such as B cells, T cells, macrophages, or polymorphonucleocytes, to stimuli such as antigens or vaccines (such as influenza vaccines). Immune responses can include any body cells involved in the host's defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate immune responses or inflammation. As used herein, a protective immune response refers to an immune response that protects a subject from infection (preventing infection or the development of infection-related disease). Methods for measuring immune responses include, for example, measuring the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc. Other examples are provided herein.

[0049] Immunize: To protect a subject (eg, a mammal) from an infectious disease (eg, influenza), such as by vaccination.

[0050] Inactivated: In the context of this disclosure, an "inactivated" virus is a virus that has been altered to the point that it cannot establish infection in a host or host cells. Viruses can be inactivated using, for example, chemicals, heat, pH alteration, and / or irradiation (such as ultraviolet or gamma irradiation). Inactivated viruses are also referred to as "killed" viruses. "Chemically inactivated" viruses are viruses inactivated using chemical methods (such as treatment with BPL, formaldehyde, glutaraldehyde, 2,2'-dithiopyridine, or binary ethylene imine). For a review of viral vaccine inactivation methods, see Delrue et al. (Expert Rev Vaccines 11(6):695-719, 2012).

[0051] Influenza virus: A segmented, negative-strand RNA virus belonging to the family Orthomyxoviridae. Influenza viruses are enveloped viruses. There are three types of influenza viruses: A, B, and C.

[0052] Influenza A virus (IAV): A segmented, negative-sense, single-stranded RNA virus with eight RNA segments (PB2, PB1, PA, NP, M, NS, HA, and NA) encoding 11 proteins, including RNA-guided RNA polymerase proteins (PB2, PB1, and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunits HA1 and HA2), matrix proteins (M1 and M2), and nonstructural proteins (NS1 and NS2). The virus is prone to rapid evolution through misfolded protein polymerases and segment reassortment. The host range of influenza A is very diverse, including humans, birds (e.g., chickens and waterfowl), horses, marine mammals, pigs, bats, mice, ferrets, cats, tigers, leopards, and dogs. Animals infected with influenza A often serve as reservoirs for influenza viruses, and some subtypes have been shown to cross the species barrier in humans.

[0053] Influenza A viruses can be divided into multiple subtypes based on allelic variations in the antigenic regions of two genes encoding surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and cellular release. There are currently 18 different influenza A virus HA antigenic subtypes (H1 to H18) and 11 different influenza A virus NA antigenic subtypes (N1 to N11). H1-H16 and N1-N9 can be found in wild avian reservoirs and may pose a pandemic threat to humans. H17-H18 and N10-N11 have been described in bat reservoirs and are not currently considered to pose a pandemic threat to humans.

[0054] Specific examples of influenza A include, but are not limited to, H1N1 (e.g., 1918 H1N1), H1N2, H1N7, H2N2 (e.g., 1957 H2N2), H2N1, H3N1, H3N2, H3N8, H4N8, H5N1, H5N2, H5N8, H5N9, H6N1, H6N2, H6N5, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H8N4, H9N2, H10N1, H10N7, H10N8, H11N1, H11N6, H12N5, H13N6, and H14N5. In one example, influenza A includes those known to circulate in humans, such as H1N1, H1N2, H3N2, H7N9, and H5N1.

[0055] In animals, most influenza A viruses cause self-limited, localized infections of the respiratory tract of mammals and / or the digestive tract of birds. However, highly pathogenic influenza A strains, such as H5N1, cause systemic infections in poultry, with mortality rates potentially reaching 100%. In 2009, H1N1 influenza was the most common cause of influenza in humans. In 2009, a new strain of H1N1 virus, originating from pigs, emerged and was declared a pandemic by the World Health Organization. This strain became known as "swine flu." The H1N1 influenza A virus also caused the 1918 Spanish flu pandemic, the 1976 Fort Dix outbreak, and the 1977-1978 Russian flu pandemic.

[0056] Influenza B virus (IBV): A negative-sense, single-stranded RNA virus with eight RNA segments. IBV has eight RNA segments (PB1, PB2, PA, HA, NP, NA, M1, and NS1) that encode 11 proteins, including RNA-guided RNA polymerase proteins (PB1, PB2, and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunits HA1 and HA2), matrix protein (M1), nonstructural proteins (NS1 and NS2), and ion channel proteins (NB and BM2). This virus is less prone to evolution than influenza A, but its mutations are sufficient to prevent the acquisition of lasting immunity. Influenza B has a narrower host range than influenza A and is only known to infect humans and seals. Influenza B viruses are not divided into subtypes, but can be further divided into lineages and strains. Specific examples of influenza B include, but are not limited to, B / Yamagata, B / Victoria, B / Shanghai / 361 / 2002, and B / Hong Kong / 330 / 2001.

[0057] Influenza C virus (ICV): A negative-sense, single-stranded RNA virus with seven RNA segments encoding nine proteins. ICV is a genus and species of the Orthomyxoviridae family. ICV infects humans and pigs, usually causing only mild symptoms, but can be severe and cause local epidemics. Unlike IAV and IBV, ICV lacks HA and NA proteins. Instead, ICV expresses a single glycoprotein called hemagglutinin-esterase fusion (HEF).

[0058] Isolated: An "isolated" biological component (e.g., a nucleic acid, protein, or virus) has been substantially separated or purified from other biological components (e.g., cell debris or other proteins or nucleic acids). Biological components that have been "isolated" include those components purified by standard purification methods. The term also encompasses recombinant nucleic acids, proteins, viruses, and chemically synthesized nucleic acids or peptides.

[0059] Neuraminidase (NA): An influenza virus membrane glycoprotein. NA participates in the destruction of the cellular receptor for the viral HA by cleaving terminal sialic acid residues from carbohydrate moieties on the surface of infected cells. NA also cleaves sialic acid residues from viral proteins, preventing viral aggregation. NA (along with HA) is one of the two major influenza virus antigenic determinants.

[0060] Outbreak: As used herein, an influenza virus "outbreak" refers to a series of virus isolates from a single country or region in a given year.

[0061] Pharmaceutically acceptable vehicles: Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, such as one or more influenza virus compositions disclosed herein, as well as other agents.

[0062] Prevent, Treat, or Ameliorate a Disease: "Preventing" a disease means inhibiting the full development of the disease. "Treatment" refers to therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after it has already begun to develop. "Amelioration" refers to reducing the number or severity of the signs or symptoms of a disease.

[0063] Purified: The term "purified" does not require absolute purity; rather, it is intended to be used as a relative term. Thus, for example, a purified protein, virus, nucleic acid, or other compound is one that is completely or partially separated from naturally associated proteins and other contaminants. In some embodiments, the term "substantially purified" refers to a protein, virus, nucleic acid, or other active compound that has been isolated from cells, cell culture medium, or other crude preparation and fractionated to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0064] Subject: A living multicellular vertebrate organism, including humans and non-human mammals such as non-human primates. In one example, a subject is a person who can be infected with influenza virus, such as humans, birds (e.g., chickens, turkeys), horses, pigs, bats, mice, ferrets, cats, tigers, leopards, seals, and dogs. As used herein, a "minor subject" is a human subject under the age of 21; an "adult subject" is a human subject 21 years of age or older.

[0065] Therapeutic effective amount: The amount of a particular drug agent sufficient to achieve the desired effect in a subject treated with that drug agent. For example, it may be an amount of influenza virus composition that can be used to induce an immune response in a subject and / or to prevent infection or disease caused by an influenza virus. In one instance, a therapeutically effective amount of influenza virus composition is an amount sufficient to enhance resistance to infection caused by an influenza virus (such as influenza A, influenza B, or both) in a subject, to prevent, improve, and / or treat infection caused by an influenza virus (such as influenza A, influenza B, or both) without causing substantial cytotoxic effects. The effective amount of influenza virus composition used to enhance resistance to infection, prevent, improve, and / or treat infection in a subject will depend on, for example, the subject being treated, the method of administration of the therapeutic composition, and other factors.

[0066] Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered to prevent, improve, or treat diseases such as infectious diseases. Immunogenic substances may include the influenza virus compositions disclosed herein. Vaccines can elicit both preventative (defensive) and therapeutic responses. Methods of administration vary depending on the vaccine but may include inoculation, oral administration, intranasal administration, intramuscular administration, or other forms of administration. Vaccines may be administered with adjuvants to enhance the immune response.

[0067] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. The singular forms “a / an” and “the” include plural indicative words. Unless the context clearly indicates otherwise, “comprising A or B” means including A or B or A and B. It should be further understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes. Although similar or identical methods and materials to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including the interpretation of terms) shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0068] III. Introduction

[0069] Influenza A virus infection is a major global public health problem. According to the World Health Organization (WHO), epidemics result in approximately 3-5 million severe cases and up to 500,000 deaths annually. Zoonotic outbreaks in which humans are infected with influenza viruses of animal origin, such as avian influenza H5N1 or H7N9 strains, can result in extremely high mortality rates and pose a significant risk of the development of new pandemic influenza strains. The impact of influenza pandemics varies, but the 1918 pandemic killed at least 50 million people worldwide and was likely derived from an avian influenza virus. The 1957 and 1968 pandemics also had surface protein genes derived from avian influenza. Unlike some infectious diseases, influenza cannot be eradicated because the causative influenza A virus is maintained through latent infection of the gastrointestinal tract of wild birds. These avian viruses form 144 possible subtypes based on the expression of one of 16 subtypes of the surface hemagglutinin (HA) molecule in combination with one of nine neuraminidase (NA) subtypes. The emergence in humans of host-adapted zoonotic influenza A viruses with novel HA (and / or NA) to which humans lack immunity is a cause of pandemics. Current public health strategies for preventing influenza are based on the annual development of new vaccines against specific circulating virus strains. This approach is inadequate, partly because the annual production and large-scale transportation of new vaccines are costly, but primarily because these annual vaccines are specific to the target circulating viruses and therefore do not protect against the emergence of new pandemic viruses with novel HA subtypes.

[0070] To address this problem, the present disclosure describes a multivalent vaccine for immunizing animals against all influenza A virus subtypes maintained in wild birds. PCT Publication WO 2015 / 195218 describes a study in which a mixture of non-infectious virus-like particles (VLPs) expressing four different low-pathogenic avian HAs (H1, H3, H5, H7) was delivered intranasally to experimental animals. The mixture induced broad reactive immunity to multiple influenza A virus subtypes (including 1918 H1N1 virus, highly pathogenic avian H5N1 and avian H7N9 virus). The VLP mixture also provides cross-protection against viruses with HA subtypes that do not appear in the vaccine (1957 H2N2 virus, and avian H6, H10, H11 and H15 viruses). Vaccination with the VLP mixture after a 10-fold lethal dose challenge between subtypes does not provide sterile immunity; however, the vaccine mixture does prevent severe cases and protect animals from death.

[0071] This disclosure describes vaccination with a whole-virus vaccine inactivated by β-propiolactone (BPL) consisting of multiple (e.g., 4, 6, or 8) different low-pathogenic avian subtypes. The BPL-inactivated influenza virus mixture, when administered intranasally or intramuscularly, provides very broad protection and heterologous protection against lethal challenges, for example, compared to VLP mixtures with the same HA.

[0072] VLP manufacturing is currently expensive, while manufacturing whole-virus inactivated vaccines is more economical and does not require the development of new manufacturing methods. Furthermore, because these vaccines consist of inactivated whole viruses, they are not as easily reassorted as live attenuated vaccines. Inactivated whole-virus vaccines can also promote antibody responses against T-cell epitopes targeting the HA head and stem regions, NA and M2e surface proteins, and other viral proteins in the inactivated virus vaccine.

[0073] IV. Overview of Implementation Methods

[0074] A universal influenza virus vaccine composition comprising multiple inactivated influenza A viruses is described. The composition comprises four or more different influenza A viruses (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 different influenza A viruses), each virus having a different HA subtype, and wherein each virus is monovalent (e.g., having only a single HA subtype, such as only H1 or only H5, rather than H1 and H5). This composition can be used, for example, to elicit an immune response against influenza viruses, to immunize a subject against seasonal influenza viruses, and / or to mitigate future pandemics as a pre-pandemic vaccine. In some instances, the composition elicits an immune response against an influenza virus subtype not present in the composition, i.e., providing a heterologous subtype immune response. For example, if the composition contains influenza viruses having subtypes H1, H3, H5, and H7, a heterologous subtype immune response may be directed against influenza viruses H2, H4, H6, H8, H9, H10, H11, H12, H13, H14, H15, and / or H16. Similarly, if the composition contains influenza viruses having subtypes H2, H4, H9, and H10, a heterologous subtype immune response may be directed against influenza viruses H1, H3, H5, H6, H7, H8, H11, H12, H13, H14, H15, and / or H16.

[0075] A composition is disclosed comprising a first influenza A virus containing a first subtype HA; a second influenza A virus containing a second subtype HA; a third influenza A virus containing a third subtype HA; and a fourth influenza A virus containing a fourth subtype HA, wherein the at least four different influenza A viruses are inactivated.

[0076] In some embodiments, at least one influenza A virus is a low pathogenicity avian influenza virus. In certain examples, at least two, at least three, or all four influenza A viruses are low pathogenicity avian influenza viruses.

[0077] In some embodiments, at least one of the first, second, third, and fourth HA subtypes is selected from H3, H4, H7, H10, H14, and H15. In some examples, at least two of the first, second, third, and fourth HA subtypes are selected from H3, H4, H7, H10, H14, and H15.

[0078] In some embodiments, at least one of the first, second, third, and fourth HA subtypes is selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. In some examples, at least two of the first, second, third, and fourth HA subtypes are selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16.

[0079] In some embodiments, at least one of the first, second, third, and fourth HA subtypes is selected from H3, H4, H7, H10, H14, and H15; and at least one of the first, second, third, and fourth HA subtypes is selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. In some examples, at least two of the first, second, third, and fourth HA subtypes are selected from H3, H4, H7, H10, H14, and H15; and at least two of the first, second, third, and fourth HA subtypes are selected from H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16.

[0080] In some examples, the first, second, third, and fourth HA subtypes are H1, H3, H5, and H7, respectively (see Figure 3 , quadrivalent vaccine 1). In specific non-limiting examples, the H1 virus is A / mallard / Ohio / 265 / 1987 (H1N9), the H3 virus is A / pintail / Ohio / 339 / 1987 (H3N8), the H5 virus is A / mallard / Maryland / 802 / 2007 (H5N1), and / or the H7 virus is A / Environment / Maryland / 261 / 2006 (H7N3). In other examples, the first, second, third, and fourth HA subtypes are H2, H4, H9, and H10 (see Figure 3, quadrivalent vaccine 2). In specific non-limiting examples, the H2 virus is A / green-winged teal / Ohio / 175 / 1986 (H2N1), the H4 virus is A / green-winged teal / Ohio / 344 / 1986 (H4N2), the H9 virus is A / Mallard / Maryland / 798 / 2007 (H9N1), and / or the H10 virus is A / Mallard / Ohio / 99 / 1989 (H10N7).

[0081] In some embodiments, the composition further comprises influenza A virus type 5, which contains the HA of subtype 5, wherein the influenza A virus type 5 is inactivated. In some examples, the composition further comprises influenza A virus type 6, which contains the HA of subtype 6, wherein the influenza A virus type 6 is inactivated. In some examples, the composition further comprises influenza A virus type 7, which contains the HA of subtype 7, wherein the influenza A virus type 7 is inactivated. In some examples, the composition further comprises influenza A virus type 8, which contains the HA of subtype 8, wherein the influenza A virus type 8 is inactivated.

[0082] From a biological systems perspective, there are two main groups of influenza A viruses (HA): Group 1 includes H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16, and Group 2 includes subtypes H3, H4, H7, H10, H14, and H15 (see [link to relevant documentation]). Figure 3 In some embodiments of the compositions disclosed herein, the composition comprises at least one influenza A virus having an HA from Group 1 and at least one influenza A virus having an HA from Group 2. In certain examples, half of the viruses (e.g., 2, 3, or 4 viruses) in the composition have HA from Group 1 and half of the viruses (e.g., 2, 3, or 4 viruses) have HA from Group 2.

[0083] In some implementations, the first, second, third, fourth, fifth, sixth, seventh, and / or eighth HA subtypes are H1, H2, H3, H4, H5, H7, H9, and H10 (e.g., Figure 3combination of quadrivalent vaccines 1 and 2 in the . In specific, non-limiting examples, the H1 virus is A / mallard / Ohio / 265 / 1987 (H1N9), the H3 virus is A / pintail / Ohio / 339 / 1987 (H3N8), the H5 virus is A / mallard / Maryland / 802 / 2007 (H5N1), the H7 virus is A / Environment / Maryland / 261 / 2006 (H7N3), the H2 virus is A / green-winged teal / Ohio / 175 / 1986 (H2N1), the H4 virus is A / green-winged teal / Ohio / 344 / 1986 (H4N2), the H9 virus is A / Mallard / Maryland / 798 / 2007 (H9N1), and / or the H10 virus is A / Mallard / Ohio / 99 / 1989 (H10N7).

[0084] The influenza A virus of the disclosed compositions is inactivated. The virus can be inactivated using any means known in the art. In some embodiments, the influenza A virus is chemically inactivated. In some instances, the influenza A virus is chemically inactivated using beta-propiolactone (BPL). In other instances, the influenza A virus is chemically inactivated using formaldehyde. In other instances, the influenza A virus is chemically inactivated using glutaraldehyde, 2,2'-dithiodipyridine, or diethyleneimine. In other embodiments, the influenza A virus is inactivated using heat. In other embodiments, the influenza A virus is inactivated using irradiation. In some instances, the influenza A virus is inactivated using ultraviolet light or gamma irradiation.

[0085] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, adjuvant, or both.

[0086] In some embodiments, the composition is formulated for intranasal administration. In other embodiments, the composition is formulated for intramuscular administration.

[0087] Also provided are containers containing the compositions disclosed herein. In some embodiments, the container is a syringe. In some instances, the syringe includes a needle. The plunger in the syringe may have a stopper to prevent the plunger from being accidentally removed during aspiration. Disposable syringes typically contain a single dose of vaccine. The syringe may have a tip cap to seal the tip before the needle is attached. In a non-limiting example, the tip cap may be made of rubber, such as butyl rubber.

[0088] In other embodiments, the container is a vial. In some instances, the vial is made of glass, such as colorless glass, for example, borosilicate glass. In other instances, the vial is made of plastic. The vial may include a stopper, such as a rubber stopper, or a cap, such as a cap adapted for syringe insertion. In some instances, the vial contains a single-dose composition. In other instances, the vial contains multiple-dose compositions, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses of the composition. Typically, the vial is sterilized before the composition is added.

[0089] Kits containing containers disclosed herein are also provided. In some embodiments, the kit includes vials (such as vials containing the composition), syringes (e.g., empty syringes or syringes containing the composition), needles, or any combination thereof. The composition may be in a suspension, such as in PBS or other pharmaceutically acceptable carriers. Alternatively, the composition may be in a dry or powder form, such as lyophilized or freeze-dried, which is subsequently reconstituted by the end user (e.g., with PBS or other pharmaceutically acceptable carriers). Containers in the kit may contain adjuvants, or adjuvants may be in separate containers within the kit. In some instances, containers may contain pharmaceutically acceptable carriers such as PBS, or pharmaceutically acceptable carriers such as PBS may be in separate containers (e.g., if the composition is lyophilized or freeze-dried). In some instances, containers in the kit further include one or more stabilizers. In some instances, the kit also includes a device that allows administration of the composition to a subject. Examples of such devices include syringes or syringe nebulizers (e.g. Nasal drug delivery devices, such as those available from Life Medicals Supplier, Sunrise, FL.) The kit can be packaged (eg, in the same box) with instructions containing details of the vaccine, such as instructions for administration and / or details of the virus within the vaccine.

[0090] Further, a method is provided for inducing an immune response against influenza virus in a subject by administering a therapeutically effective amount of the disclosed composition to the subject. A method is also provided for immunizing a subject against influenza virus by administering a therapeutically effective amount of the disclosed composition to the subject. In some embodiments, the composition is administered intramuscularly. In other embodiments, the composition is administered intranasally. In some embodiments, the subject is a mammal or bird. In other embodiments, the subject is a human.

[0091] Compositions disclosed herein can be used for the treatment (such as vaccination) of children and adults. It is currently recommended to use influenza vaccines in minors and adult subjects from 6 months of age. Therefore, in some embodiments, people are adult subjects. In other embodiments, people are minor subjects. Therefore, the age of human subjects may be less than 1 year old (such as at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months or at least 11 months), 1-5 years old (such as at least 1 year old, at least 2 years old, at least 3 years old or at least 4 years old), 5-15 years old, 15-21 years old, 21-55 years old, 55-65 years old or at least 65 years old. In specific non-limiting examples, the patient is an elderly patient, such as a patient at least 65 years old. In some instances, the subject is a hospitalized patient, medical staff, members of the armed forces, military personnel, pregnant women, chronic patients, immunodeficient patients, patients who have previously taken antiviral compounds, people with egg allergies or people traveling abroad.

[0092] V. Composition

[0093] Provided herein are compositions comprising at least 4 (such as at least 5, at least 6, at least 7 or at least 8) different low pathogenicity avian influenza viruses. Each virus in the composition comprises different HA subtypes. In some embodiments, half of the viruses (such as 2, 3 or 4 species in the virus) have the HA subtype (H1, H2, H5, H6, H8, H9, H11, H12, H13 and H16) of group 1, and half of the viruses (such as 2, 3 or 4 species in the virus) have the HA subtype (H3, H4, H7, H10, H14 and H15) of group 2. All viruses in the composition are inactivated, such as chemically inactivated, for example, with BPL chemical inactivation. Representative (non-restrictive) influenza virus strains with subtypes H1-H16 that can be used in the disclosed compositions are as shown in Table 1.

[0094] Table 1. Sources of HA genes used to produce inactivated influenza virus vaccine mixtures

[0095]

[0096]

[0097] Other exemplary (non-limiting) viral strains having any of H1-H16 that can be used in the disclosed compositions are shown in Table 2.

[0098] Table 2: Exemplary Influenza Viruses

[0099]

[0100]

[0101] A. Exemplary components of the composition

[0102] The compositions provided herein containing inactivated influenza viruses may contain other agents. In some instances, the inactivated influenza viruses are present in a pharmaceutically acceptable carrier, such as saline, buffered saline, dextrose, water, glycerol, sesame oil, ethanol, and combinations thereof. The carrier and composition may be sterile. The composition may also contain a small amount of a wetting agent, emulsifier, or pH buffer. The composition may also contain conventional pharmaceutical excipients, such as, for example, pharmaceutically acceptable salts for regulating osmotic pressure, buffers, preservatives, and the like. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, or powder. In one example, the composition is a liquid or a lyophilized or freeze-dried powder. The composition may be formulated as a suppository using conventional binders and carriers, such as triglycerides. Oral formulations may contain standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[0103] In some examples, the composition comprising inactivated influenza virus includes a pharmaceutically acceptable carrier and an adjuvant, such as a mucosal adjuvant, for example, one or more of CpG oligodeoxynucleotides, Flt3 ligand, and monophosphate formyl A (MLA). In one example, the adjuvant includes MLA, such as a clinical grade formulation, for example (3-O-deacylated-4'-monophosphoryllipid A) adjuvant.

[0104] B. Formulations for Mucosal Administration

[0105] The compositions provided herein comprising inactivated influenza viruses can be formulated for mucosal vaccination, such as intranasal administration. Mucosal vaccination can be achieved through a variety of routes, including oral, intranasal, pulmonary, rectal, and vaginal. In particular instances, it is achieved through intranasal administration. Therefore, in some instances, the disclosed compositions are formulated for intranasal administration.

[0106] For example, the disclosed compositions may include one or more biodegradable, mucoadhesive polymer carriers. Polymers such as polylactic acid-co-glycolide (PLGA), chitosan, alginate, and carbopol may be included. Hydrophilic polymers such as alginate and carbopol are absorbed into mucus by forming hydrogen bonds, thereby extending nasal residence time, and therefore may be included in the disclosed compositions.

[0107] In one example, composition includes sodium alginate, which is a linear copolymer and is composed of the α-l-guluronic acid residues of the β-d-mannuronic acid and 1-4 connections of 1-4. In some instances, composition includes Carbopol (a cross-linked polyacrylic acid polymer), for example, in combination with starch. In some instances, composition includes chitosan, a non-toxic linear polysaccharide, which can be prepared by chitin deacetylation. In one example, chitosan is the form of chitosan nanoparticles, such as N-trimethyl chitosan (TMC) base nanoparticles.

[0108] In one example, the composition is formulated into a microparticle delivery system for nasal injection. In one example, the composition comprising inactivated influenza virus can include liposomes, immunostimulatory complexes (ISCOMs) and / or polymer particles such as virosomes. In one example, the liposomes are surface-modified (e.g., coated with glycol chitosan or oligomannose). In one example, the liposomes are fusogenic or cationic membrane-fusogenic.

[0109] The composition comprising inactivated influenza virus may also comprise one or more bacterially derived lipopeptides or synthetic derivatives thereof. Examples of lipid moieties include tripalmitoyl-S-glycerocysteine ​​(Pam3Cys), dipalmitoyl-S-glycerocysteine ​​(Pam2Cys), single / multi-chain palmitic acid and lipoamino acids (LAAs).

[0110] The composition comprising inactivated influenza virus may further comprise one or more adjuvants, for example, a mucosal adjuvant, such as one or more of CpG oligodeoxynucleotides (CpG ODN), Flt3 ligand, and monophosphoryl lipid A (MLA). In one example, the adjuvant comprises a clinical grade MLA formulation, for example (3-O-deacylated 4'-monophosphoryl lipid A) adjuvant.

[0111] V. Methods for stimulating immune responses

[0112] Provided herein are methods for using the disclosed compositions comprising inactivated influenza viruses. In one example, the method includes eliciting a broad reactive immune response against influenza viruses (e.g., influenza A, influenza B, or both) in a subject. In another example, the method includes immunizing the subject against influenza viruses (e.g., influenza A, influenza B, or both) or vaccinating the subject against influenza viruses (e.g., influenza A, influenza B, or both).

[0113] For example, the disclosed inactivated influenza virus can stimulate a broadly reactive immune response, thereby protecting subjects administered the inactivated influenza virus composition from severe illness or death caused by multiple influenza A viruses (and / or influenza B viruses) without requiring matching between the challenge virus strain and the vaccine composition. This paper demonstrates that broad cross-protection (i.e., heterologous subtype protection) is achieved when the inactivated influenza virus composition does not contain the same HA subtype as the challenge virus strain (e.g., the challenge virus strain is H6N1, H10N1, or H10N7, but the composition does not contain any subtype H6 or H10 virus). Therefore, the disclosed inactivated influenza virus composition can be used as a pre-pandemic vaccine.

[0114] Therefore, in some instances, the immune response induced by the disclosed composition is against one or more of the following (e.g., at least two, at least three, at least four, or at least five): H1N1, H1N2, H1N7, H2N1, H2N2, H3N1, H3N2, H3N8, H4N8, H5N1, H5N2, H5N8, H5N9, H6N1, H6N2, H6N5, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H8N4, H9N2, H10N1, H10N7, H10N8, H11N1, H11N6, H12N5, H13N6, and H14N5. In some instances, the immune response is against one or more of H1N1, H1N2, H3N2, H7N9, and H5N1. In some instances, this immunity provides protection against attacks against one or more of the following (e.g., at least two, at least three, at least four, or at least five): H1N1, H1N2, H1N7, H2N1, H2N2, H3N1, H3N2, H3N8, H4N8, H5N1, H5N2, H5N8, H5N9, H6N1, H6N2, H6N5, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H8N4, H9N2, H10N1, H10N7, H10N8, H11N1, H11N6, H12N5, H13N6, and H14N5. In some examples, such immunity provides protection against challenge with one or more of H1N1, H1N2, H3N2, H7N9, and H5N1 (e.g., preventing infection or preventing the development of a disease associated with the infection). In one example, the inactivated influenza virus compositions disclosed herein can be used as influenza vaccines that elicit a protective immune response against H1N1 and / or H3N2 influenza viruses.

[0115] In some examples, the immune response or immunity is achieved with a mixture of inactivated influenza viruses expressing HA subtypes different from the HA subtype that achieves at least one immune response or protection. For example, if a mixture of inactivated viruses expressing H2, H3, H5, and H7 is administered to a subject, at least one immune response or immunity to an H1 influenza virus, such as H1N1, can be achieved. For example, if a mixture of inactivated viruses expressing H1, H3, H5, and H7 is administered to a subject, at least one immune response or immunity to one or more of the following: H2, H4, H6, H8, H9, H10, H11, H12, H13, H14, H15, and H16 influenza viruses can be achieved.

[0116] In some embodiments, the disclosed compositions comprising inactivated influenza viruses are administered using any suitable route of administration, such as intranasally or intramuscularly. In some embodiments, the compositions comprising inactivated influenza viruses include a pharmaceutically acceptable carrier and / or adjuvant. For example, the pharmaceutically acceptable carrier can be saline, such as sterile PBS pH 7.2-pH 7.4. For example, the adjuvant can be one or more of an immunostimulatory oligonucleotide (such as a CpG oligonucleotide), a Flt3 ligand, and monophosphoryl lipid A (MLA).

[0117] The disclosed compositions can be used to stimulate or induce an immune response against an influenza virus (such as influenza A, influenza B, or both) in a subject. In some instances, the method involves administering a therapeutically effective amount of the composition comprising inactivated influenza virus provided herein to a subject, thereby inducing an immune response against the influenza virus in the subject. Methods for determining whether an immune response has been stimulated or induced are known, and several examples are provided herein. In some instances, a positive immune response or immunity is achieved if disease reduction (e.g., less weight loss, symptom relief, or improvement in lung pathology), a decrease in viral titer, and / or protection from death is observed. Thus, in some instances, the disclosed methods and / or compositions reduce weight loss by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% (e.g., within 6–15 days after attack), for example, compared to equivalent subjects who did not receive the influenza virus composition. In some instances, the disclosed methods and / or compositions reduce the symptoms of influenza infection by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, for example, compared to equivalent subjects who did not receive the composition. In some instances, the disclosed methods and / or compositions reduce lung pathology caused by influenza infection by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, for example, compared to equivalent subjects who did not receive the composition. In some instances, the disclosed methods and / or compositions reduce lung viral titers by at least 50%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold, for example, compared to equivalent subjects who did not receive the composition. In some instances, the disclosed methods and / or compositions improve survival rates after subsequent viral challenge by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 80%, at least 90%, or at least 94%, for example, compared to equivalent subjects who did not receive the composition. In some instances, the immune response achieved using the disclosed compositions is superior to that achieved using equivalent VLPs. Therefore, in some instances, the disclosed compositions have an improved ability to stimulate or elicit an immune response compared to equivalent VLPs.

[0118] The disclosed compositions can be used to immunize or vaccinate subjects against influenza viruses, such as mammalian or avian subjects. In some instances, the method includes administering a therapeutically effective amount of the composition comprising inactivated influenza virus provided herein to the subject, thereby immunizing the subject against the influenza virus. In some instances, a positive immune response or immunization is achieved if disease mitigation (e.g., reduced weight loss, reduced symptoms, reduced lung pathology), reduced viral titer, and / or protection from death is observed.

[0119] Examples of subjects to whom the disclosed inactivated influenza virus compositions can be administered include subjects that can be (or have been) infected by influenza. Examples of such subjects include, but are not limited to, mammalian and avian subjects, such as humans, pigs, chickens, turkeys, horses, dogs, and cats. Human subjects can be adults (including the elderly) or minors.

[0120] In some embodiments of the method of eliciting an immune response or immunizing a subject, about 1 to about 25 μg of each of at least 4 different inactivated viruses in a composition is administered to the subject (e.g., intranasally or intramuscularly). In a specific example, about 1 to about 5 μg of each of at least 4 different inactivated viruses in a composition is administered to the subject (e.g., intranasally or intramuscularly), about 5 to about 20 μg of each of at least 4 different inactivated viruses in the composition, or about 10 to about 15 μg of each of at least 4 different inactivated viruses in the composition. In a specific, non-limiting example, about 1.5 μg or 15 μg of each of at least 4 different inactivated viruses in a composition is administered to the subject (e.g., intranasally or intramuscularly). However, one skilled in the art will be able to determine a therapeutically effective amount of the inactivated virus administered to the subject (e.g., an amount that provides protection against H1N1 influenza virus infection).

[0121] A. Application Method

[0122] The disclosed compositions comprising inactivated influenza viruses can be administered to a subject by any approach commonly used for introducing the virus into a subject. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, mucosal, vaginal, rectal, intranasal, inhalation, or oral administration. Parenteral administration, such as subcutaneous, intravenous, or intramuscular administration, is typically achieved by injection. Injections can be prepared in conventional forms, can be liquid solutions or suspensions, are suitable for dissolving or suspending in a solid form in a liquid before injection, or as an emulsion. Injection solutions and suspensions can be made of sterile powders, granules, tablets, etc. Administration can be systemic or local.

[0123] The composition comprising inactivated influenza virus administered to the subject is administered together with at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be determined in part by the specific composition administered and by the specific method used to administer the composition. Therefore, there are a variety of suitable formulations of the pharmaceutical compositions of the present disclosure. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, sesame oil, ethanol, and combinations thereof. The composition may also contain conventional pharmaceutical excipients, for example, pharmaceutically acceptable salts, buffers, preservatives, etc. for regulating osmotic pressure. The carrier and composition may be sterile, and the formulation may be suitable for the mode of administration. The composition may also contain a small amount of a wetting agent or an emulsifier or a pH buffer. The composition may be a liquid solution, a suspension, an emulsion, a tablet, a pill, a capsule, a sustained-release formulation, or a powder.

[0124] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include liquids and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose) etc. Preservatives and other additives may also be present, for example, antibacterial agents, antioxidants, chelating agents and inert gases etc.

[0125] Some compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid; organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid; inorganic bases, such as sodium hydroxide, ammonium hydroxide, potassium hydroxide; organic bases, such as mono-, di-, trialkyl, and arylamines, and substituted ethanolamines; or by reaction with inorganic or organic bases.

[0126] In a particular instance, the compositions provided herein are formulated for mucosal vaccination, such as oral, intranasal, pulmonary, rectal and vaginal. In a particular instance, this is achieved by intranasal administration. For example, the disclosed compositions may include one or more biodegradable, mucoadhesive polymer carriers. Polymers such as polylactic acid-co-glycolide (PLGA), chitosan (e.g., in the form of chitosan nanoparticles, such as nanoparticles of N-trimethyl chitosan (TMC)), alginates (e.g., sodium alginate) and carbitol may be included. In one example, the composition includes one or more hydrophilic polymers, such as sodium alginate or carbopol. In one example, the composition includes carbopol, for example, in combination with starch. In one example, the composition is formulated as a microparticle delivery system for nasal administration. Therefore, the composition comprising inactivated influenza virus may include liposomes, immunostimulating complexes (ISCOMs), and / or polymer particles such as viral particles. The composition comprising inactivated influenza virus may further comprise one or more bacterially derived lipopeptides or synthetic derivatives thereof, such as Pam3Cys, (Pam2Cys, single / multi-chain palmitic acid and lipoamino acids (LAA). The composition may further comprise one or more adjuvants, such as one or more of CpG oligodeoxynucleotides (CpG ODN), Flt3 ligand and monophosphoryl lipid A (MLA). In one embodiment, the adjuvant comprises a clinical grade MLA formulation, e.g. (3-O-deacylated-4'-monophosphoryllipid A) adjuvant.

[0127] B. Timing of application

[0128] The disclosed compositions comprising four or more inactivated influenza viruses are administered in a single dose or multiple doses (e.g., an enhancer). In some instances, a second administration is performed after the first administration. For example, the second administration may be performed with a composition comprising inactivated influenza virus that is the same as or different from the first composition comprising inactivated influenza virus that was administered. In a specific instance, the second administration is performed using a composition comprising inactivated influenza virus that is the same as the first composition comprising inactivated influenza virus that was administered. In another specific instance, the second administration is performed using a composition comprising inactivated influenza virus that is different from the first composition comprising inactivated influenza virus that was administered. For example, if the first composition comprising inactivated influenza virus comprises a first HA subtype, a second HA subtype, a third HA subtype, and a fourth HA subtype, the second composition comprising inactivated influenza virus may comprise a fifth HA subtype, a sixth HA subtype, a seventh HA subtype, and an eighth HA subtype, wherein all eight subtypes are different.

[0129] In some instances, compositions containing four or more inactivated influenza viruses are administered in multiple doses, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (e.g., 2-3 doses). In such instances, the time between doses may be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, or at least 10 years, such as 1-4 weeks, 2-3 weeks, 1-6 months, 2-4 months, 1-10 years, or 2-5 years, or combinations thereof (e.g., when there are at least three administrations, the time between the first and second doses and between the second and third doses may be the same or different).

[0130] C. Dosage

[0131] In the context of this disclosure, the dose administered to a subject should be sufficient to induce a beneficial therapeutic response in the subject over time, or to inhibit or prevent influenza virus infection. The required dose may vary depending on the subject's species, age, weight and general condition, the severity of the infection to be treated, the specific composition used, and the method of administration. Those skilled in the art can determine an appropriate dose using only routine testing.

[0132] In some embodiments, about 1 to about 100 μg of each of the at least 4 different inactivated viruses in the composition is administered to the subject (e.g., intranasally or intramuscularly), such as about 1 μg to about 50 μg, 1 μg to about 25 μg, 1 μg to about 5 μg, about 5 μg to about 20 μg, or about 10 μg to about 15 μg of each of the at least 4 different inactivated viruses in the composition. In a specific, non-limiting example, about 15 μg of each of the at least 4 different inactivated viruses in the composition is administered to the subject (e.g., intranasally or intramuscularly). In another specific, non-limiting example, about 10 μg of each of the at least 4 different inactivated viruses in the composition is administered to the subject (e.g., intranasally or intramuscularly). In a specific, non-limiting example, about 20 μg of each of the at least 4 different inactivated viruses in the composition is administered to the subject (e.g., intranasally or intramuscularly). In one specific, non-limiting example, about 1 μg or 2 μg of each of at least four different inactivated viruses in the composition is administered to the subject (eg, intranasally or intramuscularly).

[0133] D. Methods for Measuring Immune Responses

[0134] Methods for determining whether or not the compositions disclosed herein comprising inactivated influenza viruses can elicit or stimulate an immune response (e.g., achieving successful immunization) are known in the art. For example, see Cottey et al., in Current Contents in Immunology 19.11.1-19.11.32, 2001 (incorporated herein by reference). Although exemplary assays are provided herein, the present disclosure is not limited to the use of specific assays.

[0135] After using the inactivated influenza virus composition provided herein, one or more determinations can be performed to assess the immune response gained. In some instances, also before using the composition, determination is performed, to be used as a baseline or control. Samples, such as blood or serum samples, are collected from the subject after using the composition. In some instances, samples are collected at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks or at least 8 weeks (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks) after first use. Samples subsequently can also be obtained, for example, after subsequent use.

[0136] 1. Hemagglutination Titer Assay

[0137] Hemagglutination titers can be performed to measure or evaluate hemagglutination units (HAU). This assay can be used to evaluate whether a virus expresses a functional HA trimer and can also be used to quantify HA proteins in viral preparations. Hemagglutinin titers are also used to quantify the amount of influenza virus used as a challenge virus, or, for example, to quantify the amount of virus present in the lungs or respiratory tract of an attacked animal. Vaccinated subjects may show a decrease in viral titer compared to subjects receiving a blank vaccination.

[0138] This assay can be used to quantify the amount of virus in a sample, such as a lung sample from a subject challenged with the virus and pre-treated with the composition containing inactivated influenza virus provided herein. The virus stock solution is serially diluted (e.g., a two-fold dilution from 1:4 to 1:4096) and then added to wells containing red blood cells (RBCs). An RBC solution (e.g., 0.75% to 1% RBC) is added to the wells. The mixture is then incubated at room temperature for 30 minutes, allowing the RBCs to settle. The resulting agglutination pattern of the sample is then analyzed, for example, by examining microtiter wells in which the sample is placed. For example, in a microtiter plate placed at its edge, the RBCs in the RBC control wells will flow in a characteristic teardrop pattern (no influenza virus present, therefore no agglutination). Wells containing influenza virus will agglutinate RBCs to varying degrees. Wells with the largest amount of virus will appear cloudy because the virus cross-links all the red blood cells, preventing their precipitation. Subsequent wells with smaller amounts of virus may result in partial agglutination, but the precipitate will not flow in a teardrop shape as in the RBC control wells. The endpoint is usually determined as the maximum dilution of the virus sample that results in complete agglutination of the RBCs.

[0139] The number of HAU in the sample to be titrated can be determined. The HA titer is the reciprocal of the dilution of the last well in the series that shows complete RBC agglutination (e.g., if the last dilution is 1:640, the titer of the sample is 640 HA units / 5 μl sample).

[0140] 2. Hemagglutination inhibition (HAI) assay

[0141] In one instance, a hemagglutination inhibition (HAI) assay was performed after administration of the composition comprising inactivated influenza virus provided herein. Influenza virus can agglutinate red blood cells, a process called hemagglutination, as described above. Hemagglutination is blocked in the presence of specific antibodies against surface hemagglutinin. This phenomenon provides the basis for the HAI assay, which is used to detect and quantify specific antiviral antibodies in serum. Therefore, HAI measures the presence of antibodies that block HA receptor binding (as assessed by hemagglutination of RBCs).

[0142] In one example, serum to be evaluated for the presence of antibodies to the hemagglutinin head is treated with receptor destroying enzyme (RDE) overnight at 37°C. The following day, RDE is inactivated by incubation at 56°C for 1 hour. The assay plate used is a sterile, non-tissue culture treated 96-well round-bottom microtiter plate. Two-fold serial dilutions of each sample are made down the plate from rows B to G. 50 μl of a working dilution of viral antigen (a set number of HAU) is added to all wells of the microtiter plate, except for row H (RBC control wells) and the antigen control wells. The plate is incubated at room temperature for 30 minutes. 50 μl of a 1% RBC suspension in PBS is added to all wells, and the plate is incubated at room temperature for 30 to 45 minutes. The microtiter plate is analyzed to read the agglutination pattern. Negative control wells (those containing normal serum without anti-influenza antibodies) will appear turbid because the influenza virus has completely agglutinated the RBCs. As long as there are enough anti-influenza antibodies to inhibit agglutination, the positive control wells (those containing known anti-influenza antisera) will have an RBC precipitate that looks similar to the H row control precipitate. As the serum dilution increases, the amount of antibody will decrease so that an increase in the amount of RBC agglutination will become apparent. The hemagglutination inhibition (HAI) titer of each serum sample is the reciprocal of the maximum dilution that completely inhibits RBC agglutination (i.e., the last well in the dilution series that forms an RBC precipitate). The HAI titer of each sample is the average of the endpoint titers of its duplicate dilution series. If the titers of the duplicates differ by more than two-fold dilution, the HAI titration can be repeated for that sample.

[0143] 3. Influenza Virus Neutralization Assay

[0144] In one example, a neutralization assay is performed after administration of a composition provided herein. In this assay, serum samples from subjects who have received a composition comprising inactivated influenza virus provided herein are diluted, influenza virus is added, and the amount of serum required to prevent viral growth is determined. Neutralization assesses the presence of antibodies that inhibit viral replication. Antibodies directed against the HA stem, for example, can neutralize viral replication but will not affect hemagglutination because the epitope is not located around the receptor binding domain. Antibodies that bind to the head and inhibit hemagglutination are generally neutralizing.

[0145] In some examples, serum samples are incubated in tissue culture medium (e.g., DMEM / 5% FBS containing antibiotics), for example, in a 96-well round-bottom tissue culture treated microtiter plate. Serum samples are serially diluted, for example, in duplicate adjacent wells of a microtiter plate (e.g., initially diluted 1:10 to a sample dilution of 1:640). Previously titrated influenza virus (of any subtype) can be diluted to contain 1 TCID 50 / 50μl. Add an equal volume of working stock virus (e.g., approximately 50 TCID50)50 ) was added to each serum sample (including serial dilutions) and incubated at 37°C for 1 hour. Using this rule, if the final amount of virus is between 10 and 100 TCID 50 After incubation, the neutralization titer will be 2.5x10 5 Tissue culture medium (e.g., DMEM / 5% FBS containing antibiotics) of MDCK cells / ml (or other cells) is added to the serum sample (e.g., added to all wells of a microtiter plate). It is incubated overnight in a humidified 37°C, 5% CO2 incubator. Some influenza viruses grow better at temperatures of 34° to 35°C, so these temperatures are used in some instances. The culture medium is removed and replaced with a tissue culture medium (e.g., DMEM containing antibiotics) containing trypsin (e.g., 0.0002%), and the mixture is incubated in a humidified 37°C, 5% CO2 incubator for 4 days. Subsequently, a sterile 0.5% RBC / PBS solution is added, and the mixture is incubated at 4°C for 1 hour to check whether there is agglutination in the wells. The virus neutralization titer of a specific serum sample is defined as the reciprocal of the highest dilution of serum in which both wells do not show RBC agglutination.

[0146] Samples containing sufficient concentrations of influenza virus neutralizing antibodies (e.g., in microwells) will prevent the virus from infecting cells, so viral propagation will not occur. Adding RBCs to these wells will result in the formation of RBC precipitates. In contrast, samples without anti-influenza antibodies or with anti-influenza antibodies below the neutralizing concentration (e.g., in microwells) will have influenza virus at the end of the 4-day incubation. The RBCs added to these samples will agglutinate. The influenza virus cross-links the red blood cells, inhibiting their sedimentation in the microwells, so these wells present precipitates.

[0147] 4. Neuraminidase Inhibition (NI) Antibody Titer Determination

[0148] Neuraminidase inhibition (NI) antibody titers can also be determined. To measure NI antibody titers, reassortant viruses comprising suitable NA can be generated, for example, by using plasmid-based reverse genetics (e.g., see Sandbulte et al., Influenza Other Respir Viruses 3:233-40, 2009). Suitable NA will be the same as those present in the virus administered to the subject. Fetuin can be used as a NA substrate for NI determination (e.g., see Cate et al., Vaccine 28:2076-9, 2010, incorporated herein by reference). Exemplary methods are provided below.

[0149] The NI titer is the reciprocal of the maximum dilution of serum that provides at least 50% inhibition of NA activity. It is expected that the use of the compositions disclosed herein will reduce or even eliminate the challenge virus titer of subjects who receive the inactivated influenza virus composition. For example, compared to subjects who do not receive the inactivated influenza virus composition (e.g., recipients of the blank vaccine), it is expected that the virus in the lungs of subjects who receive the inactivated influenza virus composition will be at least 10-fold, at least 20-fold, at least 50-fold, or even 100-fold less.

[0150] NI antibody titers can be determined in an enzyme-linked lectin assay using peroxidase-labeled peanut agglutinin (PNA-PO) conjugated to desialylated fetuin. NA activity can be determined by incubating serial dilutions of purified full-length NA on fetuin-coated microtiter plates. After incubation at room temperature for 30 minutes, the plates are washed and PNA-PO is added. After incubation at room temperature for one hour, the plates are washed again and the peroxidase substrate 3,3',5,5'-tetramethylbenzidine is added and color development is performed for 10 minutes. Color development is stopped and the OD450 of the plate is measured. The dilution corresponding to 95% NA activity is determined.

[0151] NI titers for NA subtypes can be measured starting with a serum dilution of 1:20, followed by 2-fold serial dilutions in a 96-well U-bottom tissue culture plate. NA corresponding to 95% of maximum activity is added to the diluted serum and incubated at room temperature for 30 minutes, after which the serum / NA samples are transferred to a fetuin-coated microtiter plate. The plate is incubated at 37°C for 2 hours, washed, and PNA-PO is added. The plate is incubated at room temperature for another hour, washed, and the peroxidase substrate TMB is added. Color development is stopped after 10 minutes, and the OD450 of the plate is measured. The NI titer is the reciprocal dilution at which 50% NA activity is inhibited. The lower limit of quantification for this assay is 20; titers below 20 are considered negative and are assigned a value of 10. In some instances, a good or positive reaction produces a value of >30, while a poor reaction or no reaction produces a value of <20.

[0152] 5. Viral Lung Titer and Pathology

[0153] Lung titers and pathology of the virus can be determined. Tissue samples, such as lung samples (e.g., aerated lung samples), are fixed (e.g., in 10% formaldehyde for 24 hours), embedded (e.g., in paraffin), sectioned (e.g., 1 to 10 μm, e.g., 5 μm), and mounted.

[0154] The antigenic distribution of influenza virus can be evaluated by immunohistochemistry using appropriate antibodies (e.g., polyclonal or monoclonal antibodies specific to the virus used to attack the subject, or polyclonal or monoclonal antibodies that are cross-reactive with different influenza virus strains). It is anticipated that the use of the compositions disclosed herein will reduce or even eliminate viral titers in subjects receiving the inactivated influenza virus composition. For example, compared to subjects who did not receive the inactivated influenza virus composition (e.g., those who received a blank vaccine), it is anticipated that subjects receiving the inactivated influenza virus composition will have at least 10-fold, at least 20-fold, at least 50-fold, or even 100-fold less virus in their lungs. In another instance, it is anticipated that the use of the compositions disclosed herein will reduce or even eliminate symptoms of influenza infection, such as bronchitis, bronchiolitis, alveolitis, and / or pulmonary edema, in subjects receiving the composition. For example, compared with subjects who did not receive the inactivated influenza virus composition (e.g., blank vaccine recipients), subjects who received the inactivated influenza virus composition were expected to have a reduction of at least 20%, at least 50%, at least 75%, or at least 90% in bronchitis, bronchiolitis, alveolitis, and / or pulmonary edema (or such reduction in the severity of these symptoms).

[0155] 6. Other Exemplary Assays

[0156] In some instances, respiratory IgA and systemic IgG, T-cell responses in subjects were assessed. Such methods are routine (e.g., see Gauger et al., Methods Mol Biol. 1161:303-12, 2014; Larsen et al., Vet Microbiol. 74(1-2):117-31, 2000; Steitz et al., PLoS One. 5(5):e10492, 2010).

[0157] In some examples, immune responses are analyzed by transcriptomics and cytokine ELISA or other cytokine immunoassays. Such methods are routine.

[0158] In some examples, immune responses are analyzed by microneutralization. Such methods are routine (e.g., see Gauger et al., Methods Mol Biol. 1161: 313-24, 2014).

[0159] In some examples, immune responses were analyzed by anti-HA stem assays. Such methods are conventional (e.g., Wu et al., PLoS One 7(8):e42363, 2012).

[0160] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0161] Example

[0162] Example 1: Chemically inactivated influenza virus mixture as a universal influenza vaccine

[0163] This embodiment describes a study evaluating an influenza virus vaccine composed of a mixture of BPL-inactivated low-pathogenic avian influenza virus subtypes. The first vaccine mixture comprises influenza viruses having HA subtypes H1, H3, H5, and H7, and the second vaccine mixture comprises influenza viruses having HA subtypes H2, H4, H9, and H10 (see [link to documentation]). Figure 3 The influenza viruses used in the assay were monovalent (e.g., included only a single or individual HA subtype).

[0164] A quadrivalent vaccine consisting of four different BPL-inactivated whole influenza viruses (A / mallard / Ohio / 265 / 1987(H1N9), A / pintail / Ohio / 339 / 1987(H3N8), A / mallard / Maryland / 802 / 2007(H5N1) and A / Environment / Maryland / 261 / 2006(H7N3)) was compared with a virus-like particle (VLP) vaccine containing VLPs expressing HA from the same virus. Figure 1 The reciprocal HAI titers of mice vaccinated with either the quadrivalent influenza VLP vaccine or the BPL-inactivated whole-virus quadrivalent influenza virus are shown. Titers against H1N1, H3N8, H5N1, and H7N3 are also shown. The results indicate that both vaccines elicited a broad range of heterologous subtype immune responses.

[0165] The ability of a BPL-inactivated influenza virus vaccine to protect against influenza virus challenge was evaluated in mice. Mice were vaccinated intramuscularly (IM) or intranasally (IN) with the BPL-inactivated vaccine and boosted three weeks later ( Figure 2A ). 50 days after the first immunization, the vaccinated mice were challenged with H6N1, H7N1, H10N1 or H10N7 influenza viruses. Figure 2B As shown, all mice vaccinated with either IN or IM vaccine survived challenge with each influenza virus, whereas mice vaccinated with the mock vaccine died 6–8 days after challenge.

[0166] Another study was conducted to evaluate the immune response induced by a second vaccine mixture composed of four different BPL-inactivated whole influenza viruses (A / green-winged teal / Ohio / 175 / 1986 (H2N1), A / green-winged teal / Ohio / 344 / 1986 (H4N2), A / Mallard / Maryland / 798 / 2007 (H9N1), and A / Mallard / Ohio / 99 / 1989 (H10N7)). Mice were vaccinated with the H2 / H4 / H9 / H10 quadrivalent vaccine, and reciprocal HAI titers against H1N1, H6N1, H7N1, and H15N1 were measured. The results showed that the H2 / H4 / H9 / H10 quadrivalent vaccine elicited a broad range of heterologous subtype immune responses.

[0167] The ability of a BPL-inactivated H2 / H4 / H9 / H10 quadrivalent influenza virus vaccine to protect against influenza virus challenge was also evaluated in mice. Mice were vaccinated with either a blank vaccine or an H2 / H4 / H9 / H10 quadrivalent vaccine IM or IN and boosted three weeks later. Vaccinated mice were challenged with H1N1, H6N1, H7N1, or H15N1 influenza viruses 50 days after the first immunization. Vaccinated mice showed 90% survival (<0.05) against H1 and H6 challenges. Figure 4A and 4B It also shows a 100% survival rate against H7 and H15 attacks. Figure 4C and 4D Vaccination (IN or IM) also provides 100% protection against attacks of the same virus type (H10N1). Figure 4E ).

[0168] Additional studies were conducted to evaluate the immune response induced by a vaccine mixture consisting of eight different BPL-inactivated whole influenza viruses (A / mallard / Ohio / 265 / 1987(H1N9), A / pintail / Ohio / 339 / 1987(H3N8), A / mallard / Maryland / 802 / 2007(H5N1), A / Environment / Maryland / 261 / 2006(H7N3), A / green-winged teal / Ohio / 175 / 1986(H2N1), A / green-winged teal / Ohio / 344 / 1986(H4N2), A / Mallard / Maryland / 798 / 2007(H9N1) and A / Mallard / Ohio / 99 / 1989(H10N7)). Mice were vaccinated with the H1 / H3 / H5 / H7 / H2 / H4 / H9 / H10 octavalent vaccine and reciprocal HAI titers were measured against H1N1, H3N8, H5N1, and H7N3 (and / or other influenza viruses). The octavalent vaccine is expected to generate a broad, heterologous immune response.

[0169] The ability of a BPL-inactivated H1 / H3 / H5 / H7 / H2 / H4 / H9 / H10 octavalent vaccine to protect against influenza virus challenge was evaluated in mice. Mice were vaccinated with the octavalent vaccine either intramuscularly (IM) or intravenously (IN) and boosted three weeks later. Following the initial immunization, mice were challenged with H2N2, H6N1, H11N1, or H15N1 influenza viruses. Mice vaccinated with either the IN or IM vaccine were expected to survive each influenza virus challenge.

[0170] Example 2: Testing of inactivated influenza virus vaccines in ferrets

[0171] Not infected with influenza Ferrets (Mustela putorius furo, female, 6-12 months old) were purchased from Marshall Farms (Sayre, PA, USA). Ferrets were housed in pairs in stainless steel cages (Shor-line, Kansas City, KS, USA) containing Sani-chips Laboratory Animal Bedding (PJ Murphy Forest Products, Montville, NJ, USA). Ferrets were provided with Teklad Global Ferret Diet (Harlan Teklad, Madison, WI, USA) and water ad libitum.

[0172] Ferrets were vaccinated with either a blank vaccine or a quadrivalent vaccine administered IN or IM consisting of four different BPL inactivated whole influenza viruses: A / mallard / Ohio / 265 / 1987 (H1N9), A / pintail / Ohio / 339 / 1987 (H3N8), A / mallard / Maryland / 802 / 2007 (H5N1), and A / Environment / Maryland / 261 / 2006 (H7N3). Two ferrets were included in each vaccination group. Ferrets were challenged with antigenic variants and pathogenic strains A / Port Chalmers / 1971 (H3N2) and A / swine / Iowa / 1931 (H1N1) four weeks after vaccination. Vaccinated ferrets provided significant protection against challenge with both strains. In both cases, vaccinated animals showed a 10% reduction in viral nasal wash titers on day 5 compared to blank-vaccinated animals. 3 to 10 4 log( Figure 5A ), and showed significantly less lung pathology ( Figure 5B ).

[0173] In another experiment, ferrets vaccinated with the quadrivalent H1 / H3 / H5 / H7 vaccine were challenged with H2 and H10 influenza viruses to assess heterologous subtype protection in vaccinated ferrets. Ferrets vaccinated with the quadrivalent vaccine were found to lose less weight than animals vaccinated with the sham vaccine. Expectedly, viral titers in lung and nasal wash samples were also lower in vaccinated animals.

[0174] In other studies, ferrets were administered two doses of inactivated influenza virus vaccine (e.g., quadrivalent H1 / H3 / H5 / H7, quadrivalent H2 / H4 / H9 / H10, or octavalent H1 / H3 / H5 / H7 / H2 / H4 / H9 / H10) IN at week 0, followed by a booster with the same dose at week 3. During the vaccination regimen, animals were monitored weekly for adverse events, including weight loss, decreased body temperature, decreased activity, nasal discharge, sneezing, and diarrhea. Prior to vaccination, animals were confirmed to be seronegative for circulating influenza A and B viruses by HAI assay. Blood was collected from anesthetized ferrets via the anterior vena cava 14 to 21 days after each vaccination and transferred to microcentrifuge tubes. The tubes were centrifuged, the serum removed, and frozen at -80 ± 5°C. The immune response of the serum was analyzed, for example, by HAI serum antibody titers.

[0175] One to three weeks after the final vaccination, a lethal dose (10xMLD 50 Ferrets were challenged intranasally with a variety of pathogenic influenza viruses, including 1918 H1N1, 1957 H2N2, 1968 H3N2 and 2009 H1N1 pandemic viruses, highly pathogenic H5N1, H7N9, and H6N1 avian influenza viruses and other related challenge viruses. After infection, the ferrets were monitored daily for weight loss, signs of illness, and death within 14 days of infection. Individual weight, disease score, and death were recorded daily for each group after inoculation. Nasal washes were performed by instilling 3 ml of PBS into the nostrils of anesthetized ferrets every day for 7 days after inoculation. The wash was collected and stored at -80°C until use. Serum can also be collected.

[0176] Ferret survival and vaccine-induced immunity were assessed, for example, by hemagglutination inhibition, microneutralization, and anti-HA stalk assays, as well as neuraminidase inhibition assays. Vaccinated animals were also evaluated for respiratory IgA and systemic IgG, T cell responses, viral lung titers, and pathology, as well as immune responses by transcriptomics and cytokine ELISA.

[0177] It is expected that the inactivated influenza virus cocktail will protect ferrets against challenge with all of these viruses.

[0178] Example 3: Human clinical trials

[0179] After the best broadly cross-reactive inactivated influenza virus vaccine is selected in experimental animals, studies are conducted in human volunteers. In some instances, the vaccine will include an adjuvant suitable for human use.

[0180] Intranasal vaccine formulations comprising inactivated (e.g., BPL-inactivated) whole influenza viruses will be produced using GMP methods (e.g., tetravalent H1 / H3 / H5 / H7, tetravalent H2 / H4 / H9 / H10, or octavalent H1 / H3 / H5 / H7 / H2 / H4 / H9 / H10) and administered intranasally to humans. Those skilled in the art will appreciate that other inactivated influenza virus vaccine compositions provided herein can be similarly tested.

[0181] In short, the human influenza vaccine composition is administered intranasally. A booster dose of the same mixture is given to the same person approximately 3–12 weeks later (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks later). A second group of people receives a blank vaccine (e.g., with saline). Blood and nasal samples are collected and stored. Any adverse events in patients are monitored during the study. Because inactivated vaccines are non-infectious, they are expected to have excellent safety profiles.

[0182] If the vaccine composition is demonstrated to be safe in a Phase I trial, a Phase II efficacy trial is conducted using a human volunteer influenza challenge model developed by the NIH Clinical Center (e.g., see Memoli et al., Validation of a Wild-Type Influenza A Human Challenge Model: H1N1pdMIST, An A(H1N1)pdm09Dose Finding INDStudy). Participants are screened for health conditions and low titers (<1:10) against the 2009 pandemic H1N1 virus by HAI assay. Selected participants receive either an intranasal inactivated influenza virus vaccine (Group 1) or a saline blank vaccine (Group 2). They receive a booster at week 3 and then their serological titers are assessed by HAI or other assays at week 6, challenged with a dose of virus validated to induce influenza disease and shedding in >60% of participants with a pre-challenge HAI titer <1:10. Vaccine efficacy is assessed by the development of a serological response to vaccination, alleviation of symptoms, reduction in viral titers, and / or a decrease in the duration of viral shedding.

[0183] In view of the many possible embodiments to which the principles of this disclosure may be applied, it should be recognized that the illustrated embodiments are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims. Therefore, we claim as our invention all inventions that fall within the scope and spirit of these claims.

Claims

1. A composition comprising: A first influenza A virus comprising a hemagglutinin (HA) of a first subtype; a second influenza A virus comprising a HA of a second subtype; A third influenza A virus comprising a third subtype HA; and a fourth influenza A virus comprising a fourth subtype HA, wherein the first, second, third and fourth influenza A viruses are inactivated, and wherein the first, second, third and fourth HA subtypes are H1, H3, H5 and H7, or wherein the first, second, third and fourth HA subtypes are H2, H4, H9 and H10.

2. The composition of claim 1, wherein the first, second, third and fourth HA subtypes are H1, H3, H5 and H7.

3. The composition of claim 1, wherein the first, second, third and fourth HA subtypes are H2, H4, H9 and H10.

4. The composition of claim 1, further comprising a fifth influenza A virus comprising HA of a fifth subtype, wherein the fifth influenza A virus is inactivated.

5. The composition of claim 4, further comprising a sixth influenza A virus comprising HA of a sixth subtype, wherein the sixth influenza A virus is inactivated.

6. The composition of claim 5, further comprising a seventh influenza A virus comprising HA of a seventh subtype, wherein the seventh influenza A virus is inactivated.

7. The composition of claim 6, further comprising an eighth influenza A virus comprising HA of an eighth subtype, wherein the eighth influenza A virus is inactivated.

8. The composition of claim 7, wherein the first, second, third, fourth, fifth, sixth, seventh and / or eighth HA subtype is selected from H1, H2, H3, H4, H5, H7, H9 and H10.

9. The composition of any one of claims 1-8, wherein the influenza A virus is chemically inactivated.

10. The composition of claim 9, wherein the influenza A virus is chemically inactivated using beta-propiolactone (BPL).

11. The composition of any one of claims 1-8, further comprising a pharmaceutically acceptable carrier.

12. The composition of any one of claims 1-8, further comprising an adjuvant.

13. The composition of any one of claims 1-8, formulated for intranasal administration.

14. The composition of any one of claims 1-8, formulated for intramuscular administration.

15. A container comprising the composition of any one of claims 1 to 14.

16. The container of claim 15, wherein the container is a syringe or a vial.

17. Use of the composition of any one of claims 1 to 14 in the preparation of a medicament for eliciting an immune response to influenza A virus in a subject.

18. Use of the composition of any one of claims 1 to 14 in the preparation of a medicament for immunizing a subject against influenza A virus.

19. Use according to claim 17 or claim 18, wherein the medicament is suitable for intramuscular administration.

20. The use of claim 17 or claim 18, wherein the medicament is suitable for intranasal administration.

21. The use of claim 17 or claim 18, wherein the subject is a mammal or an avian.

22. The use of claim 17 or claim 18, wherein the subject is a human.

23. The use of claim 22, wherein the human is an adult subject.

24. The use of claim 22, wherein the human is a minor subject.

25. The use of claim 17 or claim 18, wherein the medicament provides a heterosubtypic immune response.

26. The use according to claim 25, wherein: The first, second, third and fourth HA subtypes are H1, H3, H5 and H7, and the heterosubtypic immune response confers at least partial protection against influenza H2, H4, H6 and / or H10 infection; The first, second, third and fourth HA subtypes are H2, H4, H9 and H10, and the heterologous subtypic immune response confers at least partial protection against influenza H1, H3, H5, H6 and / or H7 infection; The first, second, third, fourth, fifth, sixth, seventh and eighth HA subtypes are H1, H2, H3, H4, H5, H7, H9 and H10, and the heterologous subtypic immune response confers at least partial protection against influenza H6 and / or H8 infection.

27. A kit comprising a container as claimed in claim 15 or claim 16.

28. The kit of claim 27, further comprising instructions for administration of the composition and / or a description of the components of the composition.

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