WO2016118937A1 - Influenza virus vaccination regimens - Google Patents

Influenza virus vaccination regimens Download PDF

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Publication number
WO2016118937A1
WO2016118937A1 PCT/US2016/014640 US2016014640W WO2016118937A1 WO 2016118937 A1 WO2016118937 A1 WO 2016118937A1 US 2016014640 W US2016014640 W US 2016014640W WO 2016118937 A1 WO2016118937 A1 WO 2016118937A1
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Prior art keywords
influenza virus
domain
polypeptide
hemagglutinin
chimeric
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French (fr)
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Peter Palese
Adolfo Garcia-Sastre
Florian KRAMMER
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Icahn School of Medicine at Mount Sinai
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Icahn School of Medicine at Mount Sinai
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Priority to AU2016209032A priority Critical patent/AU2016209032A1/en
Priority to US15/545,548 priority patent/US10736956B2/en
Priority to JP2017538596A priority patent/JP2018504412A/ja
Priority to CA2974699A priority patent/CA2974699A1/en
Priority to EP16740886.3A priority patent/EP3247389A4/en
Publication of WO2016118937A1 publication Critical patent/WO2016118937A1/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • HHSN272201400008C and HHSN266200700010C awarded by the National Institutes of Health. The government has certain rights in the invention.
  • immunization/vaccination regimens for inducing an immune response (e.g., an antibody response) against influenza virus.
  • an immune response e.g., an antibody response
  • influenza virus e.g., an influenza virus
  • immunization regimens involve the administration of a chimeric hemagglutinin (HA), a headless HA or another influenza virus stem domain based construct (e.g., the HA stem domain or a fragment thereof) to a subject.
  • the immunization regimens also involve the administration of an influenza virus neuraminidase immunogen.
  • Influenza viruses are enveloped RNA viruses that belong to the family of
  • Orthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Viruses and Their
  • influenza A viruses are mainly avians, but influenza A viruses (including those of avian origin) also can infect and cause illness in humans and other animal hosts (bats, canines, pigs, horses, sea mammals, and mustelids).
  • influenza A virus circulating in Asia has been found in pigs in China and Indonesia and has also expanded its host range to include cats, leopards, and tigers, which generally have not been considered susceptible to influenza A (CIDRAP - Avian Influenza: Agricultural and Wildlife Considerations).
  • CIDRAP - Avian Influenza Agricultural and Wildlife Considerations
  • Influenza A and B viruses are major human pathogens, causing a respiratory disease that ranges in severity from sub-clinical infection to primary viral pneumonia which can result in death.
  • the clinical effects of infection vary with the virulence of the influenza strain and the exposure, history, age, and immune status of the host.
  • the cumulative morbidity and mortality caused by seasonal influenza is substantial due to the relatively high attack rate.
  • influenza can cause between 3-5 million cases of severe illness and up to 500,000 deaths worldwide (World Health Organization (2003) Influenza: Overview; March 2003).
  • influenza viruses infect an estimated 10-15% of the population (Glezen and Couch RB (1978) Interpandemic influenza in the Houston area, 1974-76.
  • influenza viruses are the cause of infrequent pandemics.
  • influenza A viruses can cause pandemics such as those that occurred in 1918, 1957, 1968, and 2009. Due to the lack of pre-formed immunity against the major viral antigen, hemagglutinin (HA), pandemic influenza can affect greater than 50% of the population in a single year and often causes more severe disease than epidemic influenza.
  • HA hemagglutinin
  • pandemic influenza can affect greater than 50% of the population in a single year and often causes more severe disease than epidemic influenza.
  • pandemic of 1918 in which an estimated 50-100 million people were killed (Johnson and Mueller (2002) Updating the Accounts: Global Mortality of the 1918-1920 "Spanish” Influenza Pandemic Bulletin of the History of Medicine 76: 105-115).
  • influenza viruses are constantly undergoing change: every 3-5 years the predominant strain of influenza A virus is replaced by a variant that has undergone sufficient antigenic drift to evade existing antibody responses. Isolates to be included in vaccine preparations must therefore be selected each year based on the intensive surveillance efforts of the World Health Organization (WHO) collaborating centers. Second, to allow sufficient time for vaccine manufacture and distribution, strains must be selected approximately six months prior to the initiation of the influenza season. Often, the predictions of the vaccine strain selection committee are inaccurate, resulting in a substantial drop in the efficacy of vaccination.
  • WHO World Health Organization
  • the immunization/vaccination regimens involve the use of a headless HA, chimeric HA, or another influenza virus stem domain based construct, such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix, e.g., amino acids 76-130 of A/Hong
  • the immunization/vaccination regimens involve supplementing a seasonal influenza vaccine with a headless HA, chimeric HA, or another influenza virus stem domain based construct, such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix, e.g., amino acids 76-130 of A/Hong Kong/1/1968, numbered according to the classic H3 subtype numbering system).
  • a headless HA chimeric HA
  • another influenza virus stem domain based construct such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix, e.g., amino acids 76-130 of A/Hong Kong/1/1968, numbered according to the classic H3 subtype numbering system).
  • the immunization/vaccinating regimens also involve the use of an NA immunogen. In some embodiments, the immunization/vaccinating regimens involve supplementing a seasonal influenza vaccine with NA immunogen. In certain embodiments,
  • the immunization/vaccinating regimens involve supplementing a seasonal vaccine with a fragment of NA.
  • the immunization/vaccinating regimens involve supplementing a seasonal influenza virus vaccine with an (i) NA immunogen, and (ii) a headless HA, chimeric HA, or another influenza virus stem domain based construct, such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix, e.g., amino acids 76-130 of A/Hong Kong/1/1968, numbered according to the classic H3 subtype numbering system).
  • the immunization/vaccinating regimens involve a combination of (i) a headless HA, a chimeric HA, or another influenza virus stem domain-based construct, such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix, e.g., amino acids 76-130 of A/Hong Kong/1/1968, numbered according to the classic H3 subtype numbering system), and (ii) an NA immunogen.
  • a headless HA such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix, e.g., amino acids 76-130 of A/Hong Kong/1/1968, numbered according to the classic H3 subtype numbering system)
  • an NA immunogen e.g., NA immunogen
  • the headless HA, chimeric HA, another influenza virus stem domain based construct such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix e.g., amino acids 76-130 of A/Hong Kong/1/1968, numbered according to the classic H3 subtype numbering system) and/or an NA immunogen may be administered to a subject (e.g., a human or other animal, such as a pig, horse, cow, dog, cat, and bird) in various forms, such as a live influenza viruses, inactivated influenza viruses, virus/viral-like particles ("VLPs”), subunit vaccines, split vaccines, DNA virus, polypeptides, etc.
  • a subject e.g., a human or other animal, such as a pig, horse, cow, dog, cat, and bird
  • VLPs virus/viral-like particles
  • a method for immunizing against influenza virus in a human subject comprising (a) administering to the subject a live attenuated influenza virus engineered to express a first chimeric hemagglutinin (HA), wherein the first chimeric HA comprises a first influenza virus HA globular head domain and an influenza virus HA stem domain, wherein the HA globular head domain is heterologous to the HA stem domain; and (b) a certain time after the administration of the live attenuated influenza virus, administering to the subject an inactivated influenza virus comprising a second chimeric HA, wherein the second chimeric HA comprises a second influenza virus HA globular head domain and the HA stem domain, wherein the second globular head domain is heterologous to the HA stem domain, and wherein the first HA globular head domain is different than the second HA globular head domain.
  • HA chimeric hemagglutinin
  • the method further comprises administering a neuraminidase immunogen or a vector comprising such a construct concurrently with or within 1 hour of the administration of the live attenuated influenza virus. In some aspects, the method further comprises administering an NA immunogen or a vector comprising such a construct concurrently with or within 1 hour of the administration of the inactivated influenza virus.
  • the first globular head domain comprises one or more antigenic regions from influenza virus NA. In some aspects, the second globular head domain comprises one or more antigenic regions from influenza virus NA.
  • a method for immunizing against influenza virus in a human subject comprising (a) administering to the subject a chimeric HA, a headless HA or another influenza virus stem domain based construct (e.g., the HA stem domain or a fragment thereof), or an influenza virus hemagglutinin core polypeptide or a vector comprising such a construct; and (b) subsequently administering to the subject an inactivated influenza virus vaccine, which may be supplemented with an NA immunogen(s) or a vector comprising such a construct.
  • a method for immunizing against influenza virus in a human subject comprising administering to the subject (a) an inactivated influenza virus vaccine; and (b) an NA immunogen(s) or a vector comprising such a construct.
  • a method for immunizing against influenza virus in a human subject comprising (a) administering to the subject a chimeric HA or a vector comprising such a construct; (b) subsequently administering to the subject a first headless HA or a vector comprising such a construct; and (c) subsequently administering to the subject a second headless HA or a vector comprising such a construct, wherein the first headless HA and the second headless HA are the same; wherein the chimeric HA, the first headless HA, and/or the second headless HA is administered to the subject in combination with an NA immunogen(s) or a vector comprising such a construct.
  • a method for immunizing against influenza virus in a human subject comprising (a) administering to the subject a chimeric HA or a vector comprising such a construct; (b) subsequently administering to the subject a first headless HA or a vector comprising such a construct; and (c) subsequently administering to the subject a second headless HA or a vector comprising such a construct, wherein the first headless HA and the second headless HA are different; wherein the chimeric HA, the first headless HA, and/or the second headless HA is administered to the subject in combination with an NA immunogen(s) or a vector comprising such a construct.
  • an NA immunogen is administered to a subject using a vector described herein.
  • a vector comprising a construct such as, e.g., a chimeric HA, a headless HA, or an NA immunogen, described herein is a vector as described in Section 5.8-Section 5.12.
  • a method for immunizing against influenza virus in a human subject comprising (a) administering to the subject a first headless HA or a vector comprising such a construct; and (b) subsequently administering to the subject a second headless HA or a vector comprising such a construct, wherein the first headless HA and the second headless HA are the same; and wherein the first headless HA and/or the second headless HA is administered to the subject in combination with an NA immunogen(s) or a vector comprising such a construct.
  • a method for immunizing against influenza virus in a human subject comprising (a) administering to the subject a first headless HA or a vector comprising such a construct; and (b) subsequently administering to the subject a second headless HA or a vector comprising such a construct, wherein the first headless HA and the second headless HA are different or a vector comprising such a construct; wherein the first headless HA and/or the second headless HA is administered to the subject in combination with an NA immunogen(s) or a vector comprising such a construct.
  • a vector comprising a construct such as, e.g., a chimeric HA, a headless HA, or an NA immunogen, described herein is a vector as described in Section 5.8-Section 5.12.
  • a method for immunizing against influenza virus in a human subject comprising: (a) administering to the subject a live attenuated influenza virus engineered to express a first chimeric hemagglutinin (HA), wherein the first chimeric HA comprises a first influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, wherein the HA globular head domain is heterologous to the HA stem domain; and (b) a certain time after the administration of the live attenuated influenza virus, administering to the subject an inactivated influenza virus comprising a second chimeric HA, wherein the second chimeric HA comprises a second influenza virus HA globular head domain and the HA stem domain polypeptide, wherein the second HA globular head domain is heterologous to the HA stem domain polypeptide, and wherein the first influenza virus HA globular head domain is different than the second influenza virus HA globular head domain.
  • HA chimeric hemagglutinin
  • the method further comprises administering a neuraminidase (NA) polypeptide concurrently with or within 1 hour of the administration of the inactivated influenza virus.
  • influenza virus HA stem domain polypeptide comprises an HAl N-terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HA c- term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HA c- term is the C-terminal amino acid of the HA1 domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HA1 domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HA1
  • the first and second influenza virus HA globular head domains consist of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HA1 domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HA1 domain of an H3 hemagglutinin according to H3 numbering.
  • the first influenza virus HA globular head domain comprises one or more antigenic peptides from influenza virus neuraminidase (NA).
  • the second influenza virus HA globular head domain comprises one or more antigenic peptides from influenza virus NA.
  • the antigenic region of NA is ILRTQESEC (SEQ ID NO: 107).
  • Also provided herein is a method for immunizing against influenza virus in a human subject comprising: (a) administering to the subject a live attenuated influenza virus engineered to express a chimeric HA, wherein the chimeric HA comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, wherein the HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) a certain time after the administration of the live attenuated influenza virus, administering to the subject an inactivated virus.
  • the method further comprises administering to the subject a neuraminidase (NA) polypeptide concurrently with or within 1 hour of the
  • the method further comprises administering a neuraminidase (NA) polypeptide concurrently with or within 1 hour of the administration of the inactivated influenza virus.
  • NA neuraminidase
  • influenza virus HA globular head domain consists of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of an H3 hemagglutinin according to H3 numbering.
  • influenza virus HA stem domain polypeptide comprises an HAl N-terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAc-term is the C-terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of
  • the HA globular head domain comprises one or more antigenic peptides from influenza virus neuraminidase (NA).
  • NA influenza virus neuraminidase
  • the antigenic region of NA is ILRTQESEC (SEQ ID NO: 107)
  • Also provided herein is a method for immunizing against influenza virus in a human subject, comprising: (a) administering to the subject a chimeric HA or a vector comprising such a construct, wherein the chimeric HA comprises an influenza virus HA globular head domain heterologous to the influenza virus HA stem domain polypeptide of the chimeric HA; and (b) administering to the subject an influenza virus neuraminidase polypeptide or a vector comprising such a construct.
  • influenza virus HA globular head domain consists of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • influenza virus HA stem domain comprises an HAl
  • HAl N-terminal stem segment and an HAl C-terminal stem segment
  • the HAl N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain
  • the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain
  • HA N-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide
  • HAc-term is the C-terminal amino acid of the HAl domain
  • a p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering
  • a q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of an H3 hemagglutinin according to H3 numbering.
  • a method for immunizing against influenza virus in a human subject comprising: (a) administering to the subject a first vaccine formulation comprising an influenza virus neuraminidase polypeptide and a live attenuated influenza virus engineered to express a first chimeric hemagglutinin (HA), wherein the first chimeric HA comprises a first influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) a certain time after the administration of the first vaccine formulation, administering to the subject a second vaccine formulation comprising an inactivated influenza virus comprising a second chimeric HA, wherein the second chimeric HA comprises a second influenza virus HA globular head domain and the HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide,
  • the HA stem domain polypeptide comprises an HAl N- terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N-term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N -term is the N- terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAc-term is the C- terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • the first influenza virus HA globular head domain comprises one or more antigenic peptides from influenza virus neuraminidase (NA).
  • the second influenza virus HA globular head domain comprises one or more antigenic peptides from influenza virus NA.
  • the antigenic peptide from NA is ILRTQESEC (SEQ ID NO: 107).
  • the certain time is about 3 to about 6 months after the administration of the first vaccine formulation.
  • the first and second influenza virus HA globular head domains consist of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of an H3 hemagglutinin according to H3 numbering.
  • the first influenza virus HA globular head domain comprises one or more antigenic peptides from influenza virus neuraminidase (NA).
  • the second influenza virus HA globular head domain comprises one or more antigenic peptides from influenza virus NA.
  • the antigenic peptide from NA is ILRTQESEC (SEQ ID NO: 107).
  • the certain time is about 3 to about 6 months after the administration of the first vaccine formulation.
  • the second vaccine formulation further comprises an influenza virus neuraminidase polypeptide.
  • the HA stem domain polypeptide comprises an HAl N- terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N- terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HA c- term is the C- terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys
  • influenza virus HA globular head domain comprises one or more antigenic peptides from influenza virus neuraminidase (NA).
  • NA influenza virus neuraminidase
  • the antigenic peptide from NA is ILRTQESEC (SEQ ID NO: 107).
  • the certain time is about 3 to about 6 months after the administration of the first vaccine formulation.
  • a method for immunizing against influenza virus in a human subject comprising: (a) administering to the subject a first vaccine formulation comprising a live attenuated influenza virus engineered to express a chimeric HA, wherein the chimeric HA comprises a influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) a certain time after the administration of the first vaccine formulation, administering to the subject a second vaccine formulation comprising an inactivated virus and an influenza virus neuraminidase polypeptide, wherein inactivated virus comprises a stem domain that is of the same subtype or strain as the influenza virus HA stem domain polypeptide.
  • the HA stem domain polypeptide comprises an HAl N- terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N- terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HA c- term is the C- terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • the antigenic peptide from NA is ILRTQESEC (SEQ ID NO: 107).
  • the certain time is about 3 to about 6 months after the administration of the first vaccine formulation.
  • a method for immunizing against influenza virus in a human subject comprising: (a) administering to the subject a first vaccine formulation comprising an influenza virus neuraminidase polypeptide and a first chimeric hemagglutinin (HA), wherein the first chimeric HA comprises a first influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) a certain time after the administration of the first vaccine formulation, administering to the subject a second vaccine formulation comprising a second chimeric HA, wherein the second chimeric HA comprises a second influenza virus HA globular head domain and the HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and wherein the first influenza virus HA globular head domain is different than the second influenza virus
  • the HA stem domain polypeptide comprises an HAl N- terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N- terminal amino acid of a mature HA0 protein lacking a signal peptide, wherein HA c- term is the C- terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • the first and second influenza virus HA globular head domains consist of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of an H3 hemagglutinin according to H3 numbering.
  • the second vaccine formulation further comprises an influenza virus neuraminidase polypeptide.
  • the first vaccine formulation further comprises an influenza virus neuraminidase polypeptide.
  • the one of the antigenic peptides comprises the amino acid sequence of SEQ ID NO: 107.
  • the HA stem domain polypeptide comprises an HAl N- terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N- terminal amino acid of a mature HA0 protein lacking a signal peptide, wherein HAc-term is the C- terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • the certain time is about 3 to about 6 months after the administration of the first vaccine formulation.
  • a method for immunizing against influenza virus in a human subject comprising (a) administering to the subject a first vaccine formulation comprising a first chimeric hemagglutinin (HA), wherein the first chimeric HA comprises a first influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) a certain time after the administration of the first vaccine formulation, administering to the subject a second vaccine formulation comprising an influenza virus neuraminidase polypeptide and a second chimeric HA, wherein the second chimeric HA comprises a second influenza virus HA globular head domain and the HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and wherein the first influenza virus HA globular head domain is different than the second influenza virus
  • the first and second influenza virus HA globular head domains consist of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of an H3 hemagglutinin according to H3 numbering.
  • the first and second influenza virus HA globular head domains consist of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of an H3 hemagglutinin according to H3 numbering.
  • the certain time is about 3 to about 6 months after the administration of the first vaccine formulation.
  • the HA stem domain polypeptide comprises an HAl N- terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N-term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N- terminal amino acid of a mature HA0 protein lacking a signal peptide, wherein HAc-term is the C- terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • the first and second influenza virus HA globular head domains consist of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HAl domain of an H3 hemagglutinin according to H3 numbering.
  • the certain time is about 3 to about 6 months after the administration of the first vaccine formulation.
  • the HA stem domain polypeptide comprises an HAl N- terminal stem segment and an HAl C-terminal stem segment, wherein the HAl N-terminal stem segment consists of amino acid residues HA N-term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N- term is the N- terminal amino acid of a mature HA0 protein lacking a signal peptide, wherein HAc-term is the C- terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • a method for immunizing against influenza virus in a human subject comprising: (a) administering to the subject a first vaccine formulation comprising a chimeric hemagglutinin (HA), wherein the chimeric HA comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, wherein the influenza virus HA globular head domain is heterologous to the HA stem domain
  • influenza virus HA globular head domains consist of the amino acid residues intervening A p and A q , wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus
  • the terms “about” or “approximate,” when used in reference to an amino acid position refer to the particular amino acid position in a sequence or any amino acid that is within five, four, three, two, or one residues of that amino acid position, either in an N-terminal direction or a C-terminal direction.
  • the term “about” or “approximately” when used in conjunction with a number refers to any number within 1, 5 or 10% of the referenced number. In certain embodiments, the term “about” encompasses the exact number recited.
  • amino acid sequence identity refers to the degree of identity or similarity between a pair of aligned amino acid sequences, usually expressed as a percentage. Percent identity is the percentage of amino acid residues in a candidate sequence that are identical (i.e., the amino acid residues at a given position in the alignment are the same residue) or similar (i.e., the amino acid substitution at a given position in the alignment is a conservative substitution, as discussed below), to the corresponding amino acid residue in the peptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology.
  • Sequence homology may be determined using sequence alignment techniques well-known in the art, preferably computer algorithms designed for this purpose, using the default parameters of said computer algorithms or the software packages containing them.
  • Non-limiting examples of computer algorithms and software packages incorporating such algorithms include the following.
  • the BLAST family of programs exemplify a particular, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences (e.g., Karlin & Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268 (modified as in Karlin & Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877), Altschul et al, 1990, J. Mol. Biol.
  • BESTFIT which uses the "local homology” algorithm of Smith and Waterman (Advances in Applied Mathematics, 2:482-489, 1981) to find best single region of similarity between two sequences, and which is preferable where the two sequences being compared are dissimilar in length
  • GAP which aligns two sequences by finding a "maximum similarity” according to the algorithm of
  • core polypeptide in the context of an influenza virus hemagglutinin, refers to a polypeptide segment that corresponds to a region of an influenza hemagglutinin HA2 polypeptide, i.e., core polypeptides as referred to herein do not comprise an entire influenza hemagglutinin HA2 polypeptide.
  • the term refers to a polypeptide segment that corresponds to a region of the long alpha helix region of an influenza hemagglutinin HA2 polypeptide. See Section 5.3.2, infra, and Section 5.1.1 of International Publication No. WO 2011/103453 and US Application No. 2013/0209499, which are
  • the effective amount results in a reduction in titer of influenza virus relative to an untreated subject with an influenza virus infection of approximately 1 log or more, approximately 2 logs or more, approximately 3 logs or more, approximately 4 logs or more, approximately 5 logs or more, approximately 6 logs or more, approximately 7 logs or more, approximately 8 logs or more, approximately 9 logs or more, approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8 logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to 8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6 logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8 logs, 6 to 9 logs, 7 to 8 logs, 7 to 8 logs,
  • Flu HA polypeptides include, but are not limited to, chimeric influenza virus hemagglutinin polypeptides, non-chimeric influenza virus hemagglutinin polypeptides, influenza virus hemagglutinin head domain polypeptides and influenza virus hemagglutinin stem domain polypeptides.
  • the flu HA polypeptide is a chimeric influenza virus hemagglutinin polypeptide that comprises either one or more modified glycosylation sites in the influenza virus hemagglutinin stem domain that disrupts glycan binding to the stem domain; an influenza virus hemagglutinin globular head domain comprising one or more non-naturally occurring glycosylation sites; or both.
  • HAl C-terminal short stem segment refers to a polypeptide segment that corresponds to the carboxyl-terminal portion of the stem domain of an influenza hemagglutinin HAl polypeptide.
  • an HAl C-terminal short stem segment consists of amino acid residues corresponding approximately to amino acids B q through HAl c- term of an HAl domain. Residue B q is identified in influenza A hemagglutinin polypeptides in Fig. 14. Exemplary HAl C-terminal short stem segments are described herein.
  • an infection means the invasion by, multiplication and/or presence of a virus in a cell or a subject.
  • an infection is an "active" infection, i.e., one in which the virus is replicating in a cell or a subject.
  • Such an infection is characterized by the spread of the virus to other cells, tissues, and/or organs, from the cells, tissues, and/or organs initially infected by the virus.
  • An infection may also be a latent infection, i.e., one in which the virus is not replicating.
  • an infection refers to the pathological state resulting from the presence of the virus in a cell or a subject, or by the invasion of a cell or subject by the virus.
  • MOI multiplicity of infection
  • neuraminidase and “NA” encompass neuraminidase polypeptides that are modified by post-translational processing such as disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage and lipid modification (e.g. S-palmitoylation).
  • post-translational processing such as disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage and lipid modification (e.g. S-palmitoylation).
  • influenza virus subtype is an HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18 subtype.
  • the non- chimeric influenza virus hemagglutinin polypeptide comprises an HA stem domain and HA globular head domain from the same influenza virus strain.
  • the influenza virus strain is A/Netherlands/602/2009.
  • the non-naturally occurring glycosylation site has the amino acid motif Asn-Xaa-Ser/Thr/Cys, wherein Xaa is any amino acid, or, in certain embodiments, wherein Xaa is any amino acid except Pro.
  • nucleic acid is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid can be single-stranded or double- stranded.
  • the terms "purified” and “isolated” when used in the context of a polypeptide (including an antibody) that is obtained from a natural source, e.g., cells refers to a polypeptide which is substantially free of contaminating materials from the natural source, e.g., soil particles, minerals, chemicals from the environment, and/or cellular materials from the natural source, such as but not limited to cell debris, cell wall materials, membranes, organelles, the bulk of the nucleic acids, carbohydrates, proteins, and/or lipids present in cells.
  • a flu HA polypeptide e.g., an influenza hemagglutinin stem domain polypeptide, an influenza hemagglutinin head domain polypeptide, a chimeric influenza hemagglutinin polypeptide and/or a non-chimeric influenza hemagglutinin polypeptide
  • an influenza hemagglutinin stem domain polypeptide e.g., an influenza hemagglutinin stem domain polypeptide, an influenza hemagglutinin head domain polypeptide, a chimeric influenza hemagglutinin polypeptide and/or a non-chimeric influenza hemagglutinin polypeptide
  • an influenza hemagglutinin head domain polypeptide e.g., an influenza hemagglutinin head domain polypeptide, a chimeric influenza hemagglutinin polypeptide and/or a non-chimeric influenza hemagglutinin polypeptide
  • hemagglutinin stem domain polypeptide an influenza hemagglutinin head domain polypeptide, non-chimeric HA polypeptide, and/or a chimeric influenza hemagglutinin polypeptide is isolated.
  • replication refers to one or more, or all, of the stages of a viral life cycle which result in the propagation of virus.
  • the steps of a viral life cycle include, but are not limited to, virus attachment to the host cell surface, penetration or entry of the host cell (e.g., through receptor mediated endocytosis or membrane fusion), uncoating (the process whereby the viral capsid is removed and degraded by viral enzymes or host enzymes thus releasing the viral genomic nucleic acid), genome replication, synthesis of viral messenger RNA (mRNA), viral protein synthesis, and assembly of viral ribonucleoprotein complexes for genome replication, assembly of virus particles, post-translational modification of the viral proteins, and release from the host cell by lysis or budding and acquisition of a phospholipid envelope which contains embedded viral glycoproteins.
  • replication refers to the replication
  • a stem domain polypeptide is derived from an influenza hemagglutinin. In specific embodiments, a stem domain polypeptide is derived from an HI or H3 influenza virus hemagglutinin.
  • an influenza virus HA stem domain typically comprises an HA1 N-terminal stem segment and an HA1 C-terminal stem segment, wherein the HA1 N-terminal stem segment consists of amino acid residues HA N- term through A p of an influenza virus hemagglutinin HAl domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues A q through HAc-term of an influenza virus hemagglutinin HAl domain, wherein HA N-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAc-term is the C-terminal amino acid of the HAl domain, wherein A p is Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HAl domain according to H3 numbering, and wherein A q is Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HA
  • the terms “therapies” and “therapy” can refer to any protocol(s), method(s), compound(s), composition(s), formulation(s), and/or agent(s) that can be used in the prevention or treatment of a viral infection or a disease or symptom associated therewith.
  • the terms “therapies” and “therapy” refer to biological therapy, supportive therapy, and/or other therapies useful in treatment or prevention of a viral infection or a disease or symptom associated therewith known to one of skill in the art.
  • the term “therapy” refers to (i) a nucleic acid encoding a flu HA polypeptide (e.g., an chimeric influenza virus hemagglutinin polypeptide), (ii) a flu HA polypeptide (e.g., chimeric influenza virus hemagglutinin polypeptide), (iii) a vector or composition comprising a nucleic acid encoding a flu HA polypeptide (e.g., chimeric influenza virus hemagglutinin polypeptide) or comprising a flu HA polypeptide, (iv) a nucleic acid encoding an NA immunogen, (v) an NA immunogen, or (vi) a vector or composition comprising a nucleic acid encoding an NA immunogen or comprising an NA immunogen.
  • the term “therapy” refers to an antibody that specifically binds to a chimeric influenza virus hemagglutinin polypeptide.
  • the terms “treat,” “treatment,” and “treating” refer in the context of administration of a therapy(ies) to a subject to treat an influenza virus disease or infection to obtain a beneficial or therapeutic effect of a therapy or a combination of therapies.
  • Membranes were blotted with 4A5 (monoclonal antibody specific for Cal09 NA). Each panel represents a separately run Western blot of a unique vaccine brand. Dilutions of recombinant, baculovirus-expressed Cal09 rNl (shown on the left blot in every panel) of known
  • Fig. 7B Quantities of Nl NA (in ⁇ g) per adult vaccine dose (0.5 mL) as measured by ELISA. Bar graphs show the mean quantification and standard errors of the means (SEM), with mean values displayed above each corresponding bar.
  • cH5/lCal09NlCal09 inactivated vaccine (cH5/l IIV).
  • the cH8/l LAIV - cH5/l IIV group was boosted with a live attenuated vaccine based on cH8/lCal09NlCal09 virus (cH8/l LAIV) and was then also boosted again with cH5/l IIV.
  • Control animals received mock booster vaccination (prime-only group) or were vaccinated with regular trivalent inactivated influenza virus vaccine (TIV group). Anti-Nl titers were then measured after the respective vaccinations via an endpoint titer ELISA.
  • FIG. 11 A Poultry isolation units (Plas-Labs, Lansing, MI) that were modified with a perforated plexiglass divider that separates directly infected ferrets from the immunized aerosol contact ferrets. The arrow indicates directional air flow across the plexiglass divider.
  • Fig. 1 IB Schematic of the design of the transmission experiment. The direct infected ferret was housed on the left site of the cage separated from the control and stalk vaccinated animals by a perforated divider that allowed for air flow (as indicated by dashed arrows) but prevented direct contact of the animals.
  • FIG. 15A depicts a
  • Figure 18 Minimal binding concentration of mAb 4A5 to divergent Nl NAs.
  • 4A5 binds to avian Nl s from H5N1 and H7N1 as well as to human pre-pandemic and pandemic H1N1 isolates.
  • the pandemic H1N1 viruses tested included the H1N1 components of the vaccines tested in Fig. 7.
  • A/California/07/09 was a component of Fluzone and FluLaval
  • A/Brisbane/10/10 was a component of Flucelvax
  • A/Christchurch/16/10 was a component of Fluvirin.
  • the dotted line indicates the 4A5 concentration used for ELISA quantification in Fig. 7 (3 ug/ml).
  • the headless HA, chimeric HA, another influenza virus stem domain based construct such as an influenza virus HA stem domain or a fragment of the stem domain of an influenza virus HA (e.g., the long alpha helix e.g., amino acids 76-130 of A/Hong Kong/1/1968, numbered according to the classic H3 subtype numbering system) and/or an NA immunogen may be administered to a subject (e.g., a human or other animal, such as a pig, horse, cow, dog, cat, and bird) in various forms, such as a live influenza viruses, inactivated influenza viruses, virus/viral-like particles ("VLPs”), subunit vaccines, split vaccines, DNA virus, polypeptides, etc.
  • a subject e.g., a human or other animal, such as a pig, horse, cow, dog, cat, and bird
  • a subject e.g., a human or other animal, such as a pig, horse, cow, dog, cat
  • an NA immunogen is an influenza virus NA from group 1 (e.g., Nl, N4, N5 or N8) or a fragment thereof.
  • an NA immunogen is an influenza vims NA from group 2 (e.g., N2, N3, N6, N7 or N9) or fragment thereof.
  • an NA immunogen is an influenza B virus NA or a fragment thereof.
  • an NA immunogen is a fusion protein comprising an influenza virus NA or a fragment thereof.
  • an NA immunogen is a soluble influenza virus NA protein.
  • an NA immunogen is a soluble influenza virus NA protein with N-terminal tetramerization domains and, optionally, a hexahistidine-tag(s).
  • an NA immunogen is part of a viral vector, such as an influenza virus.
  • the NA immunogen may be present naturally on the viral vector, or the viral vector may be engineered to express the NA immunogen.
  • an NA immunogen is not a part of a viral vector.
  • the cysteine residues identified as Ap and Aq in Fig. 14 be maintained since they contribute to the stability of the HA stalk domain.
  • the HA globular head domain of one influenza virus HA is swapped as a whole (between the Ap and Aq cysteine residues as shown in Fig. 14) with the HA globular head domain of heterologous influenza virus HA to maintain stability of resulting the chimeric HA since conformationally it would be closest to the native structure.
  • an influenza virus HA globular head domain of a chimeric HA comprising a deletion of one, two or more antigenic region (e.g., a region of the globular head domain known to comprise or consist of an epitope).
  • a region of the globular head domain known to comprise or consist of an epitope e.g., a region of the globular head domain known to comprise or consist of an epitope.
  • an influenza HA globular head domain of a chimeric HA comprises a non-antigenic polypeptide sequence(s) (e.g., a polypeptide sequence that is known to not induce an immune response or is known to generate an immune response that is not specific to influenza) in place of one or more of the antigenic regions (e.g., a region of the head domain known to comprise or consist of an epitope) associated with the influenza virus globular head domain.
  • the influenza virus HA globular head domain of a chimeric HA contains additional or modified glycosylation sites, such as described in International Publication No. WO 2013/043729 and U.S. Patent Application No. 14/345,816, which published as U.S. Patent Publication No. 20150132330, which are incorporated herein in their entirety.
  • influenza virus HA stem domain of a chimeric HA is a stem domain described in International Publication No. WO 2013/043729 (e.g., a stem domain described in Sections 3, 5.3, and/or 6 of International Publication No. WO 2013/043729) or in International Publication No. WO 2014/099931 (e.g., a stem domain described in Sections 3, 5.1, and/or 6 of International Publication No. WO 2013/043729) or in International Publication No. WO 2014/099931 (e.g., a stem domain described in Sections 3, 5.1, and/or 6 of International Publication No. WO
  • the HA stem domain of a chimeric HA is the stem domain of an influenza A virus HI or H3, or the stem domain of an influenza B virus.
  • the influenza virus HA stem domain of a chimeric HA is deglycosylated, such as, e.g., described in International Publication No. WO 2013/043729 and U.S. Patent Application No. 14/345,816, which published as U.S. Patent Publication No. 20150132330, which are incorporated herein in their entirety, and/or using deglycosylation techniques known in the art (e.g., deglycosylation agents).
  • Nucleic acids, and methods for producing and expressing chimeric HA, headless HA, and other influenza virus stem domain based constructs are described in U.S. Patent Application Publication No. 201002971"/ '4, U.S. Patent Application Publication No. 20130129761, U.S. Patent Application No. 14/345,816, which published as U.S. Patent Publication No. 20150132330, International Publication No. WO 2013/043729 International Publication No. WO 2013/043729, U.S. Patent Application
  • the stem domain is formed by two segments of the HA1 domain and most or all of the HA2 domain.
  • the two segments of the HA1 domain are separated, in primary sequence, by the globular head domain (see, e.g., the amino acid residues between the residues designated A p and A q in Fig. 14).
  • the chimeric influenza virus hemagglutinin polypeptides described herein maintain such a structure.
  • a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide described herein (see, e.g., Sections 5.3 and 5.4.1, infra) or in International Publication Nos. WO 2010/117786, WO 201 1/123495, WO 2013/043729, and WO
  • influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI, H2, or H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.
  • a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/Brisbane/59/2007-like (HI) and (ii) an influenza virus hemagglutinin head domain polypeptide from an influenza A virus of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide.
  • a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/California/1/1988 (H3) and (ii) an influenza virus hemagglutinin head domain polypeptide from an influenza A virus of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide.
  • a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/ Ann Arbor/6/60 (H2) and (ii) an influenza virus hemagglutinin head domain polypeptide from an influenza A virus of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus
  • the stem domain of a cH5/l chimeric influenza hemagglutinin polypeptide is the stem domain of A/California/4/2009 (H1N1) HA (or the stem domain of an A/California/4/2009-like influenza virus HA) and the globular head domain of the cH5/l chimeric influenza hemagglutinin polypeptide is the globular head domain of A/Vietnam/1203/2004 (H5) HA.
  • A/California/4/2009 (HlNl) HA (or the stem domain of an A/California/4/2009-like influenza virus HA) and the globular head domain of the cH5/l chimeric influenza hemagglutinin polypeptide is the globular head domain of A/turkey/Turkey/1/2005 (H5) HA.
  • the stem domain of a cH5/l chimeric influenza hemagglutinin polypeptide is the stem domain of A/California/4/2009 (HlNl) HA (or the stem domain of an
  • the stem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of the cH5/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of A/turkey/Turkey/1/2005 (H5) HA.
  • the stem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/harbor
  • the stem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/Indiana/10/2011 (H3N2) HA and the globular head domain of the cH5/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of A/Indonesia/5/2005 (H5) HA.
  • the stem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/Indiana/10/2011 (H3N2) HA and the globular head domain of the cH5/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of A/whooperswan/Mongolia/244/2005 (H5) HA.
  • the stem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/Victoria/361/2011 (H3N2) HA and the globular head domain of the cH7/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of A/mallard/ Alberta/24/2001 (H7) HA.
  • the stem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of the cH7/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of
  • the stem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain of the cH7/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of A/Canada/444/04 (H7) HA.
  • the stem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain of the cH7/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of
  • the stem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain of the cH7/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of
  • the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptide is the stem domain of B Florida/4/2006 HA.
  • the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptide is the stem domain of B Florida/4/2006 HA and the globular head domain of the cH5/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/Vietnam/1203/2004 (H5) HA.
  • the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptide is the stem domain of B Florida/4/2006 HA and the globular head domain of the cH5/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/Indonesia/5/2005 (H5) HA.
  • the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Florida/4/2006 HA and the globular head domain of the cH5/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/turkey/Turkey/1/2005 (H5) HA.
  • the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Brisbane/60/2008 HA and the globular head domain of the cH5/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/whooperswan/Mongolia/244/2005 (H5) HA.
  • the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA and the globular head domain of the cH7/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/chicken/Jalisco/CPAl/2012 (H7) HA.
  • the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA and the globular head domain of the cH7/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/mallard/ Alberta/24/2001 (H7) HA.
  • the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA and the globular head domain of the cH7/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/mallard/Netherlands/12/2000 (H7) HA.
  • the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Wisconsin/1/2010 HA and the globular head domain of the cH7/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/mallard/ Alberta/24/2001 (H7) HA.
  • the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Brisbane/60/2008 HA and the globular head domain of the cH7/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/Canada/504/04 (H7) HA.
  • the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptide is the stem domain of B/Brisbane/60/2008 HA and the globular head domain of the cH7/B chimeric influenza hemagglutinin polypeptide is the globular head domain of A/mallard/Netherlands/12/2000 (H7) HA.
  • the stem domain of a cB/B chimeric influenza hemagglutinin polypeptide is the stem domain of B Florida/4/2006 HA.
  • the stem domain of a cB/B chimeric influenza hemagglutinin polypeptide is the stem domain of B Florida/4/2006 HA and the globular head domain of the cB/B chimeric influenza hemagglutinin polypeptide is the globular head domain of B/Lee/1940 HA.
  • the stem domain of a cH4/3 chimeric influenza hemagglutinin polypeptide is the stem domain of A/Perth/16/09 HA (or the stem domain of an A/Perth/ 16/09-like influenza virus HA) and the globular head domain of the cH4/3 chimeric influenza hemagglutinin polypeptide is the globular head domain of A/duck/Czech/56 (or the globular head domain of an A/duck/Czech/56-like influenza virus HA).
  • the HAl N-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein ends at A p +i, A p+2 , A p+3 , A p+ ; A p+5 , A p+6 , A p+7 , A p+8 , p+9, Ap+io, Ap+ii, Ap+i 2 , Ap+i 3 , A p +i 4; A p +i5, ⁇ ⁇ + ⁇ 6 , A p +i 7 , A p +i 8 , A p +i9 ; A p+2 o, A p+2 i, A p+22 , A p+23 , A p+24j A p+2 5, A p+26 , A p+27 , A p+28 , A p+2 9 ; A p+3 o, A p+3 i, A p
  • an HAl N-terminal stem segment ending at A p+38 would end at Arg90 of an H3 hemagglutinin.
  • the end of an HAl N-terminal stem segment should be selected in conjunction with the end of the HAl C-terminal stem segment and the influenza hemagglutinin head domain polypeptide so that the resulting chimeric influenza virus hemagglutinin polypeptide is capable of forming a three-dimensional structure similar to a wild- type influenza hemagglutinin.
  • the HAl C-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein does not start at Aq (e.g., Cys 277 of an HAl subunit from an H3 hemagglutinin (i.e., according to H3 numbering)), but at a residue in sequence and structural vicinity to A q .
  • the HAl C-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein starts in the range of A q- i to A q-5 , A q-5 to A q- i 0 , A q- i 0 to A q- i5, A q- i5 to A q-20 , A q-20 to A q-25 , A q-25 to A q-30 , A q-30 to A q-35 , A q-35 to A q-40 , A q-40 to A q-45 , A q- 45 to A q-50 , A q-50 to A q-55 , A q-55 to A q-60 , A q-60 to A q-65 , A q-65 to A q-70 , A q-75 to A q-80 .
  • hemagglutinin polypeptide starts hemagglutinin amino acid position 200 (using H3 numbering); and the heterologous head domain begins at amino acid position 91 and ends at amino acid position 199 (using H3 numbering).
  • the end of the N-terminal stem segment of a chimeric influenza virus hemagglutinin polypeptide described herein is A p-1
  • the start of the C- terminal stem segment of a chimeric influenza virus hemagglutinin polypeptide described herein is A q- i
  • the end of the N-terminal stem segment of a chimeric influenza virus hemagglutinin polypeptide described herein is A p-2
  • the start of the C-terminal stem segment of a chimeric influenza virus hemagglutinin polypeptide described herein is A q-2.
  • the end of the N-terminal stem segment of a chimeric influenza virus hemagglutinin polypeptide described herein is A p-3
  • the start of the C-terminal stem segment of a chimeric influenza virus hemagglutinin polypeptide described herein is A q-3
  • hemagglutinin polypeptide described herein is A p-4
  • the start of the C-terminal stem segment of a chimeric influenza virus hemagglutinin polypeptide described herein is A q-4
  • the chimeric HA1 subunit comprises 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids of the HA1 subunit of a first influenza virus strain or subtype and the remainder of amino acids of the chimeric HA1 subunit are from a second influenza virus strain or subtype.
  • the amino acids from the first influenza virus strain or subtype can be consecutive, or can represent portions of the N- and/or C-termini of a chimeric HA1 domain.
  • the chimeric HA1 subunit comprises an influenza virus hemagglutinin head domain polypeptide comprising amino acids of two or more different subtypes or strains of influenza virus.
  • the chimeric HA1 subunit comprises a globular head with amino acids of two or more different subtypes or strains of influenza virus.
  • a chimeric influenza hemagglutinin (HA) polypeptide described herein may be conjugated to heterologous proteins, e.g., a major histocompatibility complex (MHC) with or without heat shock proteins (e.g., HsplO, Hsp20, Hsp30, Hsp40, Hsp60, Hsp70, Hsp90, or HsplOO).
  • MHC major histocompatibility complex
  • HsplO heat shock proteins
  • chimeric influenza hemagglutinin (HA) polypeptide described herein may be conjugated to proteins which stimulate the innate immune system such as interferon type 1, alpha, beta, or gamma interferon, colony stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-l, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, tumor necrosis factor (TNF)-p, T Fa, B7.1, B7.2, 4-1BB, CD40 ligand (CD40L), and drug-inducible CD40 (iCD40).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL interleukin
  • IL-2 interleukin
  • IL-4 interleukin
  • IL-6 interleukin-6
  • IL-7 IL-12
  • influenza hemagglutinin head domain polypeptides comprising amino acids from two or more strains or subtypes of influenza virus.
  • a chimeric HAl subunit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 60, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 75, 76, 77, 78, 79, or 80 amino acids of the HAl subunit of a first influenza virus strain or subtype and the remainder of amino acids of the chimeric HAl subunit are from a second influenza virus strain or subtype.
  • influenza hemagglutinin head domain polypeptides comprising deleted forms of a known influenza hemagglutinin head domain, wherein up to about 150, 145, 140, 135, 130, 125, 120, 1 15, 1 10, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from the head domain.
  • influenza HA globular head domain of a chimeric HA comprises one, two, three, or more antigenic regions from the HA of a different influenza virus strain or subtype (e.g., an influenza virus strain or subtype to which all or part of the population is naive).
  • influenza HA globular head domain of a chimeric HA comprises one, two, three, or more antigenic regions from an influenza virus NA of the same or a different subtype as the globular head domain or stem domain of the chimeric HA.
  • the one, two, three or more NA antigenic regions may replace one, two, three or more HA antigenic regions.
  • the influenza HA globular head domain of a chimeric HA comprises the amino acid sequence ILRTQESEC, which is located between residues 222 and 230 (N2 numbering) in the enzymatic active site of NA.
  • this amino acid sequence replaces one, two, three or more antigenic regions of the HA globular head domain of a chimeric HA.
  • the amino acid sequence may replace one, two, three or more of antigenic sites A, B, C, and D, wherein the globular head domain is from subtype H3.
  • the amino acid sequence may replace one, two, three or more of antigenic sites Sa, Sb, Ca and Cb, wherein the globular head domain is from subtype HI .
  • influenza hemagglutinin head domain polypeptides comprising altered forms of a known influenza hemagglutinin head domain, wherein up to about 80, 75, 70 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues of the head domain are substituted (e.g., conservatively substituted) with other amino acids.
  • influenza hemagglutinin head domain polypeptides comprising altered forms of a known influenza hemagglutinin head domain, wherein up to about 1-10, 10- 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acid residues of the head domain are substituted (e.g., conservatively substituted) with other amino acids.
  • an influenza hemagglutinin head domain polypeptide comprising a deletion of two antigenic region ⁇ e.g., two regions of the head domain known to comprise or consist of an epitope).
  • an influenza hemagglutinin head domain polypeptide comprising a deletion of three antigenic region ⁇ e.g., three regions of the head domain known to comprise or consist of an epitope).
  • influenza hemagglutinin head domain polypeptides are based on the head domain of an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and H18.
  • influenza hemagglutinin head domain polypeptides are based on the head domain of an influenza B hemagglutinin (e.g. , the head domain of the hemagglutinin of an influenza B virus described in Section 5.4, infra).
  • influenza hemagglutinin stem domain polypeptides can be assessed using techniques known in the art, such as sensitivity of the hemagglutinin molecules to trypsin digestion, as described in, e.g., Thoennes et al., 2008, Virology 370: 403-414.
  • an influenza hemagglutinin stem domain polypeptide provided herein is monomeric. In certain embodiments, an influenza hemagglutinin stem domain polypeptide provided herein is multimeric. In certain embodiments, an influenza hemagglutinin stem domain polypeptide provided herein is trimeric. Those of skill in the art will recognize that native influenza hemagglutinin polypeptides are capable of trimerization in vivo and that certain influenza hemagglutinin stem domain polypeptides provided herein are capable of trimerization. In particular embodiments described below, influenza hemagglutinin stem domain polypeptides provided herein comprise trimerization domains to facilitate trimerization.
  • influenza hemagglutinin stem domain polypeptides comprise an HA2 stem domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% amino acid sequence identity to an influenza HA2 stem domain known to those of skill in the art.
  • Exemplary known HA2 stem domains from known influenza A and influenza B hemagglutinins are provided in the tables disclosed in International Publication No. WO 2010/117786, WO 2011/123495, and WO 2013/043729, U.S. Publication Nos. 2010/0297174, 2013/0129761, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 20150132330 which are
  • influenza hemagglutinin stem domain polypeptides comprising deleted forms of HA2 stem domains wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from either or both termini of the HA2 stem domain.
  • influenza hemagglutinin stem domain polypeptides comprising altered forms of HA2 stem domains wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are conservatively substituted with other amino acids. Further provided are influenza hemagglutinin stem domain polypeptides comprising deleted and altered HA2 stem domains.
  • the influenza hemagglutinin stem domain polypeptides comprises an HA2 stem domain comprising one or more modified glycosylation sites, wherein the modified glycosylation site comprises a modification of a naturally occurring glycosylation site that disrupts the ability of a glycan to attach to the modified glycosylation site, as described in Section 5.4.1, infra.
  • the modified glycosylation site comprises a modification of a naturally occurring glycosylation site that disrupts the ability of a glycan to attach to the modified glycosylation site, as described in Section 5.4.1, infra.
  • immunogenicity and accessibility antigenic regions within the stem domain can be increased by modifying one or more glycosylation sites within the stem domain in a manner that disrupts the glycosylation (i.e. the attachment of a glycan) at the sites.
  • the primary structure of an influenza hemagglutinin stem domain polypeptide comprises, in the following order: an HAl N-terminal stem segment, a linker, an HAl C-terminal stem segment and an HA2.
  • the HAl N-terminal stem segment might be any HAl N-terminal stem segment recognized by one of skill in the art based on the definition provided herein.
  • an HAl N-terminal stem segment corresponds to a polypeptide consisting of the N-terminal amino acid of a mature HAl (i.e. an HAl lacking a signal peptide) through the cysteine residue located in sequence at approximately the 52 nd residue of the HAl .
  • the HAl N-terminal stem segment does not end exactly at A p (e.g., Cys 52 of an HAl subunit from an H3 hemagglutinin (i.e., according to H3
  • the HAl N-terminal stem segment ends at A p-1 , A p-2 , A p-3 , A p-4 , A p-5 , A p-6 , A p-7 , A p-8 , A p- 9, Ap-io, A p- ii, A p- i 2 , A p- i 3 , A p- i 4 , A p- i5, A p- i6, A p- i 7 , A p- i 8 , A p- i9, A p-2 o, A p-2 i, A p-22 , A p-23 , A p-23 , A p-24 , A p-25 , A p-26 , A p-27 , A p-28 , A p-29 , A p-30 .
  • the HAl C-terminal stem segment might be any HAl C-terminal stem segment recognized by one of skill in the art based on the definition provided herein.
  • an HAl C-terminal stem segment corresponds to a polypeptide consisting of the cysteine residue located in sequence at approximately the 277 th residue of an HAl (using H3 numbering) through the C- terminal amino acid of the HAl .
  • This cysteine residue termed A q herein, is generally capable of forming a disulfide bridge with cysteine residue A p in the N-terminal stem segment of HAl . Sequences of 17 representative influenza A hemagglutinins are presented in Fig. 14, and residue A q is identified in each.
  • the HAl C-terminal stem segment starts at about A q- i, A q-2 , A q-3 , A q-4 , A q-5 , A q-6 , A q-7 , A q-8 , A q-9 , A q- io, Aq-ii, A q- i 2 , A q- i 3 , A q- i 4 , A q- i5, A q-2 o, A q-2 5, A q-3 o, A q-3 5, A q-4 o, A q-4 5, A q- 5o, A q- 55, A q-6 o, A q-6 5, A q-7 o, A q-
  • the HAl C- terminal stem segment of the flu hemagglutinin polypeptides described herein starts in the range of Aq+i to A q+3 , A q+3 to A q+ 5, A q+5 to A q+8 , A q+8 to Aq+io, A q+ i 0 to Aq + i5, or A q+ i 5 to A q+20 .
  • the end of an HAl N-terminal stem segment should be selected in conjunction with the start of the HAl C-terminal stem segment and the linker so that the resulting HAl stem domain is capable of forming a three-dimensional structure similar, as described below, to an influenza hemagglutinin.
  • the end of the N-terminal stem segment is A p +i, and the start of the C-terminal stem segment is A q- i
  • the end of the N-terminal stem segment is A p+2
  • the start of the C-terminal stem segment is A q-2.
  • the end of the N-terminal stem segment is A p+3
  • the start of the C-terminal stem segment is A q-3.
  • the end of the N-terminal stem segment is A p+4
  • the start of the C-terminal stem segment is A q-4.
  • the end of the N- terminal stem segment is A p+5
  • the start of the C-terminal stem segment is A q-5
  • influenza hemagglutinin stem domain polypeptides comprising deleted forms of HAl C-terminal stem segments wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from either or both termini of the HAl C-terminal stem segment.
  • influenza hemagglutinin stem domain polypeptides that comprise expanded forms of HAl C-terminal stem segments wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues are added to the N-terminus of the HAl C-terminal stem segments; these added residues might be derived from the amino acid sequence of a globular head domain adjacent to an HAl C-terminal stem segment.
  • the chimeric of the stem domain of the HAl subunit comprises 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids of the stem domain of the HAl subunit of a first influenza virus strain or subtype and the remainder of amino acids of the chimeric of the stem domain of the HAl subunit are from a second influenza virus strain or subtype.
  • the influenza hemagglutinin stem domain polypeptides provided herein comprise an HA2 subunit and a chimeric of the stem domain of the HAl subunit.
  • the influenza hemagglutinin stem domain polypeptide comprises a chimeric/hybrid of the stem domain of an HAl subunit in which one or more naturally occurring glycosylation sites have been modified such that the modification, disrupts the ability of a glycan to attach to the modified glycosylation site, as described in Section 5.4.1, infra.
  • immunogenicity and accessibility antigenic regions within the stem domain can be increased by modifying one or more glycosylation sites within the stem domain in a manner that disrupts the glycosylation (i.e. the attachment of a glycan) at the sites.
  • the HAl N-terminal stem segments are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and H18.
  • the HAl N-terminal stem segment is or is based on the HA-1 N-terminal stem segment of an Ann Arbor/6/60, A/Puerto Rico/8/34, or A/Perth/ 16/2009 influenza virus.
  • the HA2 stem domain is or is based on a later discovered HA2 stem domain.
  • the signal peptide might be based on any influenza virus signal peptide known to those of skill in the art.
  • the signal peptides are based on influenza A signal peptides.
  • the signal peptides are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15 and H16.
  • the signal peptide might be any signal peptide deemed useful to one of skill in the art.
  • the luminal domain might be based on any influenza luminal domain known to those of skill in the art.
  • the luminal domain might be based on any influenza luminal domain known to those of skill in the art.
  • the transmembrane domain might be based on any influenza transmembrane domain known to those of skill in the art.
  • the transmembrane domains are based on influenza A transmembrane domains.
  • the HA2 transmembrane domains are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, and H18.
  • one or more amino acid residues in a glycosylation site are substituted with any amino acid residue that disrupts the glycosylation site.
  • one or more asparagine residues in a glycosylation sequence is substituted with alanine.
  • the asparagine at position 38 of an H3 hemagglutinin is changed to an alanine.
  • one or more of the glycosylation sites in the hemagglutinin stem domain are modified by using a chemical (e.g., a deglycosylation agent), such that glycosylation at these sites will not occur during processing and maturation of the peptide.
  • the hemagglutinin stem domain comprises one or more modified glycosylation sites as discussed in Section 5.4.1, infra.
  • the HA stem domain is as disclosed in International
  • an influenza B virus HAl N-terminal stem segment starts at residue 1 (based on numbering of an influenza B virus HAl subunit as in PDB file 3BT6) and ends at Arg 50 . In certain embodiments, an influenza B virus HAl N-terminal stem segment starts at residue 1 and ends at Ala 6 6. In some embodiments, an influenza B virus HAl N-terminal stem segment starts at residue 1 and ends at Lys 80 . In some embodiments, an influenza B virus N- terminal stem segment starts at residue 1 and ends at Arg 80 . In some embodiments, an influenza B virus N-terminal stem segment starts at residue 1 and ends at Cys54. In some embodiments, an influenza B virus N-terminal stem segment starts at residue 1 and ends at CyS9 4 . In some embodiments, an influenza B vims N-terminal stem segment starts at residue 1 and ends at
  • influenza hemagglutinin stem domain polypeptide is a hybrid polypeptide that comprises or consists essentially of segments and/or domains from a plurality of influenza strains or subtypes.
  • an influenza hemagglutinin stem domain polypeptide might comprise HAl N-terminal and HAl C-terminal stem segments from different influenza A vims HA subtypes.
  • the HAl N-terminal stem segment is from influenza A vims while the HAl C-terminal stem segment is from influenza B vims.
  • the linker is a peptide that comprises one amino acid residue, two or fewer amino acid residues, three or fewer amino acid residues, four or fewer amino acid residues, five or fewer amino acid residues, ten or fewer amino acid residues, 15 or fewer amino acid residues, 20 or fewer amino acid residues, 30 or fewer amino acid residues, 40 or fewer amino acid residues, or 50 or fewer amino acid residues.
  • the linker peptide comprises 50 or more amino acid residues.
  • the linker substantially lacks a globular head domain.
  • the linker is a direct bond.
  • the linker is selected from the group consisting of Gly, Gly-Gly, Gly-Gly- Gly, Gly-Gly-Gly and Gly-Gly-Gly-Gly-Gly.
  • the linker is selected from the group consisting of Gly-Pro and Pro-Gly.
  • the linker is a 281 turn loop, e.g.
  • influenza hemagglutinin stem domain polypeptides are capable of forming a three dimensional structure that is similar to the three dimensional structure of the stem domain of a native influenza hemagglutinin.
  • Structural similarity might be evaluated based on any technique deemed suitable by those of skill in the art. For instance, reaction, e.g. under non-denaturing conditions, of an influenza hemagglutinin stem domain polypeptide with a neutralizing antibody or antiserum that recognizes a native influenza hemagglutinin might indicate structural similarity.
  • Useful neutralizing antibodies or antisera are described in, e.g. Sui, et al, 2009, Nat. Struct. Mol. Biol. 16(3):265-273, Ekiert et al, February 26, 2009, Science
  • the antibody or antiserum is an antibody or antiserum that reacts with a non-contiguous epitope ⁇ i.e., not contiguous in primary sequence) that is formed by the tertiary or quaternary structure of a hemagglutinin.
  • the RMS deviation between the structures of a influenza hemagglutinin stem domain polypeptide and corresponding portions of a known influenza A virus hemagglutinin stem domain is 5 A or less, 4 A or less, 3 A or less, 2.5 A or less, 2 A or less, 1.5 A or less, 1 A or less, 0.75 A or less, 0.5 A or less, 0.3 A or less, 0.2 A or less, or 0.1 A or less.
  • Commercially available or open source software might be used to perform the structural superimpositions and/or RMS deviation calculations.
  • Useful examples include but are not limited to Pymol (Delano Scientific LLC), Insightll and Quanta (both from Accelrys), MIDAS (University of California, San Francisco), SwissPDB viewer (Swiss Institute of Bioinformatics), TOPOFIT (Northeastern University), CBSU LOOPP
  • any influenza hemagglutinin stem domain polypeptide provided herein can further comprise one or more polypeptide domains deemed suitable to those of skill in the art.
  • Useful polypeptide domains include domains that facilitate purification, folding and cleavage of portions of a polypeptide.
  • a His tag (His-His-His-His-His-His- His, SEQ ID NO: 101), FLAG epitope or other purification tag can facilitate purification of a polypeptide provided herein.
  • the His tag has the sequence, (His)n, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater.
  • a foldon, or trimerization, domain from bacteriophage T4 fibritin can facilitate trimerization of polypeptides provided herein.
  • the trimerization domain comprises a wildtype
  • influenza hemagglutinin stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain.
  • influenza hemagglutinin stem domain polypeptides consisting of a signal peptide covalently linked to an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain.
  • the cleavage site is a cleavage site recognized by Tobacco Etch Virus (TEV) protease (e.g., amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln- (Gly/Ser) (SEQ ID NO:50).
  • TSV Tobacco Etch Virus
  • the trimerization domain is a foldon domain.
  • the trimerization domain comprises a wildtype GCN4pII trimerization heptad repeat or a modified GCN4pII trimerization heptad repeat that allows for the formation of trimeric or tetrameric coiled coils.
  • influenza hemagglutinin stem domain polypeptides consisting of an HA1 N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is covalently linked to, in sequence, a cleavage site, a trimerization domain and a purification tag.
  • the cleavage site is a cleavage site recognized by Tobacco Etch Virus (TEV) protease (e.g., amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) (SEQ ID NO:50).
  • TSV Tobacco Etch Virus
  • the trimerization domain is a foldon domain.
  • the trimerization domain comprises a wildtype GCN4pII trimerization heptad repeat or a modified GCN4pII trimerization heptad repeat that allows for the formation of trimeric or tetrameric coiled coils.
  • the purification tag is a His tag, having the sequence, (His)n, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater.
  • influenza hemagglutinin stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal stem segment in binding association with an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain that is in turn covalently linked to an HA2 cytoplasmic domain.
  • influenza hemagglutinin polypeptides described herein are not recognized by the antibody CR6261, CR6325, CR6329, CR6307, CR6323, 2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, H98, CI 79 (produced by hybridoma FERM BP- 4517; clones sold by Takara Bio, Inc. (Otsu, Shiga, Japan)), ADC (FERM BP-4516), any other antibody described in Ekiert DC et al. (2009) Antibody Recognition of a Highly conserveed Influenza Virus Epitope. Science (published in Science Express February 26, 2009); Kashyap et al. (2008), or any other similar antibodies.
  • the typical primary structure of an influenza hemagglutinin short stem domain polypeptide comprises, in the following order: an HAl N-terminal stem segment, a linker, an HAl C-terminal short stem segment and an HA2.
  • the primary sequence can be formed by a single polypeptide, or it can be formed by multiple polypeptides. Typically, a single polypeptide is expressed by any technique deemed suitable by one of skill in the art. In single polypeptide embodiments, the HAl segments and the HA2 are in tertiary association.
  • an influenza hemagglutinin short stem domain polypeptide comprises a signal peptide.
  • the signal peptide is cleaved during or after polypeptide expression and translation to yield a mature influenza hemagglutinin short stem domain polypeptide.
  • the signal peptide can be advantageous for expression of the influenza hemagglutinin short stem domain polypeptides.
  • also provided herein are mature influenza hemagglutinin short stem domain polypeptides that lack a signal peptide.
  • the HAl C-terminal short stem segment does not start at
  • B q -10 B q -ii, B q- i 2 , B q- i 3 , B q- i 4 , B q- 15, B q-2 o, B q-2 5, B q-3 o, B q-3 5, B q-4 o, B q-4 5, B q- 50, B q- 55, B q-6 0, B q-6 5,
  • the HAl C-terminal short stem segment starts at B q+ i, B q+2 , B q+3 , B q+4 , B q+5 , B q+6 , B q+7 , B q+8 , B q+9 , or B q+ io.
  • the end of an HAl N-terminal stem segment should be selected in conjunction with the start of the HAl C-terminal short stem segment and the linker so that the resulting HAl stem domain is capable of forming a three- dimensional structure similar, as described below, to an influenza hemagglutinin.
  • influenza hemagglutinin short stem domain polypeptides comprise an HAl C-terminal short stem segment having at least 70%, 75%, 80%, 85%), 90%), 95%o, 96%) or 98%> amino acid sequence identity to an influenza HAl C-terminal short stem segment known to those of skill in the art.
  • Exemplary known HAl C-terminal short stem segments are provided in the tables disclosed in International Publication No. WO
  • the end of the N-terminal stem segment is A p-1 , and the start of the C-terminal short stem segment is B q-1.
  • the end of the N- terminal stem segment is A p-2 , and the start of the C-terminal short stem segment is B q-2
  • the end of the N-terminal stem segment is A p-3 , and the start of the C- terminal short stem segment is B q-3
  • the end of the N-terminal stem segment is A p-4
  • the start of the C-terminal short stem segment is B q-4.
  • the end of the N-terminal stem segment is A p+ i, and the start of the C-terminal short stem segment is B q +i. In certain embodiments, the end of the N- terminal stem segment is A p+2 , and the start of the C-terminal short stem segment is B q+2. In certain embodiments, the end of the N-terminal stem segment is A p+3 , and the start of the C- terminal short stem segment is B q+3 In certain embodiments, the end of the N-terminal stem segment is A p+4 , and the start of the C-terminal short stem segment is B q+4. In certain embodiments,
  • the end of the N-terminal stem segment is A p-1 , and the start of the C-terminal short stem segment is B q+1. In certain embodiments, the end of the N- terminal stem segment is A p-2 , and the start of the C-terminal short stem segment is B q+2. In certain embodiments, the end of the N-terminal stem segment is A p-3 , and the start of the C- terminal short stem segment is B q+3 In certain embodiments, the end of the N-terminal stem segment is A p-4 , and the start of the C-terminal short stem segment is B q+4 In certain embodiments,
  • influenza hemagglutinin short stem domain polypeptides comprising deleted forms of HAl C-terminal short stem segments wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from either or both termini of the HAl C-terminal short stem segment.
  • influenza hemagglutinin short stem domain polypeptides comprising deleted forms of HAl C-terminal short stem segments wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from either or both termini of the HAl C-terminal short stem segment.
  • influenza hemagglutinin short stem domain polypeptides comprising deleted forms of HAl C-terminal short stem segments wherein up to 100, 95, 90, 85, 80, 75,
  • polypeptides that comprise expanded forms of HAl C-terminal short stem segments wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues are added to the N-terminus of the HAl C-terminal short stem segments.
  • one residue is added to the C-terminal short stem segment
  • two residues are added to the C-terminal short stem segment
  • two residues are added to the N-terminal stem segment
  • three residues are added to the C-terminal short stem segment, then three residues are added to the N-terminal stem segment.
  • influenza hemagglutinin short stem domain polypeptides comprising altered forms of HAl C-terminal short stem segments wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are conservatively substituted with other amino acids.
  • influenza hemagglutinin short stem domain polypeptides comprising deleted and altered HAl C-terminal short stem segments.
  • influenza hemagglutinin short stem domain polypeptides can be based on (i.e. can have sequence identity, as described above) any influenza hemagglutinin known to those of skill or later discovered.
  • influenza hemagglutinin short stem domain polypeptides are based on an influenza A hemagglutinin.
  • influenza hemagglutinin short stem domain polypeptides are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, and H18.
  • influenza hemagglutinin short stem domain polypeptides are based on an influenza B hemagglutinin, as described in detail below.
  • the HAl C-terminal short stem segments can be based on (i.e. can have sequence identity, as described above) any HAl C-terminal short stem segments known to those of skill or later discovered.
  • the HAl C-terminal short stem segments are based on influenza A HAl C-terminal short stem segments.
  • the HAl C-terminal short stem segments are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and HI 8.
  • the HA2 stem domains can be based on (i.e. can have sequence identity, as described above) any HA2 stem domains known to those of skill, later discovered or described herein.
  • the HA2 stem domains are based on influenza A HA2 stem domains.
  • the HA2 stem domains are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, and H18.
  • a linker covalently connects the HAl N-terminal stem segment to the HAl C-terminal short stem segment.
  • the linker can be any linker deemed suitable by one of skill in the art including, but not limited to, those linkers described herein.
  • the linker is a globular head, or a fragment thereof, from an influenza virus heterologous to the influenza stem domain.
  • any influenza hemagglutinin short stem domain polypeptide provided herein can further comprise one or more polypeptide domains deemed suitable to those of skill in the art.
  • Useful polypeptide domains include domains that facilitate purification, folding and cleavage of portions of a polypeptide.
  • a His tag His-His- His-His-His-His, SEQ ID NO: 101
  • FLAG epitope or other purification tag can facilitate purification of a polypeptide provided herein.
  • the purification tag is a His tag, having the sequence, (His)n, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater.
  • trimerization domain including a foldon from bacteriophage T4 fibritin can facilitate trimerization of polypeptides provided herein.
  • the trimerization domain comprises a wildtype GCN4pII trimerization heptad repeat or a modified GCN4pII trimerization heptad repeat that allows for the formation of trimeric or tetrameric coiled coils. See, e.g., Weldon et al., 2010, PLoSO E 5(9): el2466.
  • the foldon domain can have any foldon sequence known to those of skill in the art (see, e.g., Papanikolopoulou et al, 2004, J. Biol.
  • influenza hemagglutinin short stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment in binding association with an HA2 stem domain that is covalently linked to an HA2 luminal domain.
  • influenza hemagglutinin short stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain.
  • influenza hemagglutinin short stem domain polypeptides consisting of a signal peptide covalently linked to an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to, in sequence, a cleavage site, a trimerization domain and a purification tag.
  • the protease cleavage site is a thrombin cleavage site.
  • the cleavage site has the amino acid sequence LVPRGSP (SEQ ID NO: 103).
  • the cleavage site is a cleavage site recognized by Tobacco Etch Virus (TEV) protease (e.g., amino acid sequence Glu-Asn-Leu-Tyr- Phe-Gln-(Gly/Ser) (SEQ ID NO: 50).
  • TSV Tobacco Etch Virus
  • the trimerization domain is a foldon domain.
  • the trimerization domain comprises a wildtype GCN4pII trimerization heptad repeat or a modified GCN4pII trimerization heptad repeat that allows for the formation of trimeric or tetrameric coiled coils.
  • the purification tag is a His tag, having the sequence, (Hi s)n, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater.
  • influenza hemagglutinin short stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is covalently linked to, in sequence, a cleavage site, a trimerization domain and a purification tag.
  • influenza hemagglutinin short stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment in binding association with an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain.
  • influenza hemagglutinin short stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain.
  • influenza hemagglutinin short stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain.
  • influenza hemagglutinin short stem domain polypeptides consisting of an HAl N-terminal stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal short stem segment in binding association with an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain that is in turn covalently linked to an HA2 cytoplasmic domain.
  • influenza hemagglutinin long stem domain polypeptide is an influenza hemagglutinin long stem domain polypeptide as described in
  • influenza hemagglutinin stem domain polypeptide is an influenza hemagglutinin long stem domain polypeptide.
  • the typical primary structure of an influenza hemagglutinin long stem domain polypeptide provided herein comprises, in the following order: an HA1 N-terminal long stem segment, a linker, an HA1 C- terminal long stem segment and an HA2.
  • the primary sequence can be formed by a single polypeptide, or it can be formed by multiple polypeptides.
  • a single polypeptide is expressed by any technique deemed suitable by one of skill in the art.
  • the HA1 segments and the HA2 are in tertiary association.
  • a single HA polypeptide can be cleaved, for example by a protease, under appropriate expression conditions to yield two polypeptides in quaternary association. The cleavage is typically between the HA1 C-terminal short stem segment and the HA2.
  • provided herein are multiple polypeptides.
  • the HA1 segments and HA2 are in quaternary association.
  • influenza hemagglutinin long stem domain polypeptides that comprise an HA2 stem domain, an HA2 luminal domain, an HA2 transmembrane domain and an HA2 cytoplasmic domain.
  • influenza hemagglutinin long stem domain polypeptides that comprise an HA2 stem domain, an HA2 luminal domain, and an HA2 transmembrane domain but lack some or all of the typical cytoplasmic domain.
  • influenza influenza hemagglutinin long stem domain polypeptides that comprise an HA2 stem domain, an HA2 luminal domain, and an HA2 transmembrane domain but lack some or all of the typical cytoplasmic domain.
  • influenza hemagglutinin long stem domain polypeptides that comprise an HA2 stem domain and an HA2 luminal domain but lack both an HA2 transmembrane domain and an HA2 cytoplasmic domain.
  • influenza hemagglutinin long stem domain polypeptides that comprise an HA2 stem domain but lack an HA2 luminal domain, an HA2 transmembrane domain and an HA2 cytoplasmic domain.
  • the influenza hemagglutinin long stem domain polypeptides comprise an HA2 stem domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% amino acid sequence identity to an influenza HA2 stem domain known to those of skill in the art.
  • Exemplary known HA2 stem domains from known influenza A hemagglutinins are provided in International Publication Nos. WO
  • influenza hemagglutinin long stem domain polypeptides comprising deleted forms of HA2 stem domains wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from either or both termini of the HA2 stem domain.
  • influenza hemagglutinin long stem domain polypeptides comprising altered forms of HA2 stem domains wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are conservatively substituted with other amino acids.
  • influenza hemagglutinin long stem domain polypeptides comprising deleted and altered HA2 stem domains.
  • the HAl N-terminal long stem segment can be any HAl N-terminal long stem segment recognized by one of skill in the art based on the definition provided herein.
  • an HAl N-terminal long stem segment corresponds to a polypeptide consisting of the N-terminal amino acid of a mature HAl (i.e. an HAl lacking a signal peptide) through the cysteine residue located in sequence at approximately the 97 th residue of the HAl (using H3 numbering).
  • This cysteine residue termed C p herein, is generally capable of being linked to a cysteine residue C q in the C-terminal long stem segment of HAl .
  • Sequences of 17 representative influenza A hemagglutinins are presented in Fig. 14, and residue C p is identified in each.
  • the HAl N-terminal long stem segment does not end exactly at C p (e.g., Cys 97 of an HAl subunit from an H3 hemagglutinin (i.e., according to H3 numbering)), but at a residue in sequence and structural vicinity to C p .
  • the HAl N-terminal long stem segment ends at C p- i, C p . 2 , C p . 3 , or C p . 4 .
  • the HAl N-terminal long stem segment ends at C p+1 , C p +2, C p +3, C p +4 or C p+5 .
  • influenza hemagglutinin long stem domain polypeptides comprise an HAl N-terminal long stem segment having at least 70%, 75%, 80%, 85%), 90%), 95%), 96% or 98% amino acid sequence identity to an influenza HAl N-terminal long stem segment known to those of skill in the art.
  • Exemplary known HAl N-terminal long stem segments are provided in the tables disclosed in International Publication No. WO 2010/117786, WO 2011/123495, and WO 2013/043729, U.S. Publication Nos. 2010/0297174, 2013/0129761, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No.
  • polypeptides that comprise expanded forms of HAl N-terminal long stem segments wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues are added to the C-terminus of the HAl N-terminal long stem segments; these added residues can be derived from the amino acid sequence of a globular head domain adjacent to an HAl N-terminal long stem segment.
  • influenza hemagglutinin long stem domain polypeptides comprising altered forms of HAl N- terminal long stem segments wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are conservatively substituted with other amino acids.
  • influenza hemagglutinin long stem domain polypeptides comprising deleted and altered HAl N-terminal long stem segments.
  • the HAl C-terminal long stem segment does not start at
  • C q (e.g., Ala 25 3 of an HAl subunit from an H3 hemagglutinin (i.e., according to H3 numbering)), but at a residue in sequence and structural vicinity to C q .
  • the HAl C-terminal long stem segment starts at C q- i, C q-2 , C q- 3, or C q- 4.
  • the HAl C-terminal long stem segment starts at C q+ i, C q+2 , C q +3, C q +4 or C q+5 .
  • HAl N-terminal long stem segment should be selected in conjunction with the start of the HAl C-terminal long stem segment and the linker so that the resulting HAl stem domain is capable of forming a three-dimensional structure similar, as described below, to an influenza hemagglutinin.
  • influenza hemagglutinin long stem domain polypeptides comprise an HAl C-terminal long stem segment having at least 70%, 75%, 80%, 85%), 90%), 95%o, 96%) or 98%> amino acid sequence identity to an influenza HAl C-terminal long stem segment known to those of skill in the art.
  • Exemplary known HAl C-terminal long stem segments are provided in the tables disclosed in International Publication No. WO 2010/117786, WO 2011/123495, and WO 2013/043729, U.S. Publication Nos. 2010/0297174, 2013/0129761, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 20150132330 which are incorporated herein by reference in their entireties.
  • the end of the N-terminal long stem segment is C p- i, and the start of the C-terminal long stem segment is C q- i.
  • the end of the N- terminal long stem segment is A p-2 , and the start of the C-terminal long stem segment is C q-2.
  • the end of the N-terminal long stem segment is C p-3 , and the start of the C- terminal long stem segment is C q-3.
  • the end of the N-terminal long stem segment is C p- 4, and the start of the C-terminal long stem segment is C q- 4.
  • the end of the N-terminal long stem segment is C p- i, and the start of the C-terminal long stem segment is C q +i.
  • the end of the N- terminal long stem segment is C p-2 , and the start of the C-terminal long stem segment is C q+2.
  • the end of the N-terminal long stem segment is C p-3 , and the start of the C- terminal long stem segment is C q+3.
  • the end of the N-terminal long stem segment is C p- 4, and the start of the C-terminal long stem segment is C q +4.
  • the end of the N-terminal long stem segment is C p- 5
  • the start of the C-terminal long stem segment is C q +5.
  • the end of the N-terminal long stem segment is C p +i, and the start of the C-terminal long stem segment is C q- i.
  • the end of the N-terminal long stem segment is C p+2 , and the start of the C-terminal long stem segment is C q-2.
  • the end of the N-terminal long stem segment is C p+3 , and the start of the C-terminal long stem segment is C q-3.
  • the end of the N-terminal long stem segment is C p+4 , and the start of the C-terminal long stem segment is C q-4.
  • the end of the N-terminal long stem segment is C p+5 , and the start of the C- terminal long stem segment is C q- 5.
  • influenza hemagglutinin long stem domain also provided herein are influenza hemagglutinin long stem domain
  • polypeptides comprising deleted forms of HA1 C-terminal long stem segments wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted from either or both termini of the HA1 C-terminal long stem segment.
  • one residue is added to the C-terminal long stem segment, then one residue is added to the N-terminal long stem segment; if two residues are added to the C-terminal long stem segment, then two residues are added to the N-terminal long stem segment; if three residues are added to the C-terminal long stem segment, then three residues are added to the N-terminal long stem segment.
  • influenza hemagglutinin long stem domain polypeptides comprising altered forms of HA1 C-terminal long stem segments wherein up to 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are conservatively substituted with other amino acids.
  • influenza hemagglutinin long stem domain polypeptides comprising deleted and altered HA1 C- terminal long stem segments.
  • influenza hemagglutinin long stem domain polypeptides can be based on
  • influenza hemagglutinin long stem domain polypeptides are based on an influenza A hemagglutinin.
  • influenza hemagglutinin long stem domain polypeptides are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, and H17, and H18.
  • influenza hemagglutinin long stem domain polypeptides are based on an influenza B hemagglutinin, as described in detail below.
  • the HAl N-terminal long stem segments can be based on (i.e. can have sequence identity, as described above) any HAl N-terminal long stem segments known to those of skill or later discovered.
  • the HAl N-terminal long stem segments are based on influenza A HAl N-terminal long stem segments.
  • the HAl N- terminal long stem segments are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, and H17, and H18.
  • the HAl C-terminal long stem segments can be based on (i.e. can have sequence identity, as described above) any HAl C-terminal long stem segments known to those of skill or later discovered.
  • the HAl C-terminal long stem segments are based on influenza A HAl C-terminal long stem segments.
  • the HAl C-terminal long stem segments are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, and H17, and H18.
  • the HA2 stem domains can be based on (i.e. can have sequence identity, as described above) any HA2 stem domains known to those of skill, later discovered, or described herein.
  • the HA2 stem domains are based on influenza A HA2 stem domains.
  • the HA2 stem domains are based on an influenza A hemagglutinin selected from the group consisting of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, and H17, and H18.
  • the signal peptide can be based on any influenza signal peptide known to those of skill in the art or described herein.
  • the transmembrane domain can be based on any influenza transmembrane domain known to those of skill in the art or described herein.
  • the cytoplasmic domain can be based on any influenza cytoplasmic domain known to those of skill in the art or described herein.
  • one or more of the glycosylation sites in the hemagglutinin stem domain are modified (e..g, by amino acid addition, deletion or substitution) such that glycosylation at these sites will not occur during processing and maturation of the polypeptide.
  • influenza HA typically comprises one or more glycosylation sites (e.g.
  • Xaa is any amino acid other, or, in certain embodiments, wherein Xaa is any amino acid except Pro).
  • one or more amino acid residues in a glycosylation site are conservatively substituted with an amino acid residue that disrupts the glycosylation site.
  • one or more amino acid residues in a glycosylation site are substituted with any amino acid residue that disrupts the glycosylation sequence.
  • one or more asparagine residues in a glycosylation sequence is substituted with alanine.
  • the asparagine at position 38 of an H3 hemagglutinin is changed to an alanine.
  • the hemagglutinin stem domain comprises one or more modified glycosylation sites as discussed in Section 5.4.1, infra.
  • influenza virus hemagglutinin long stem domain polypeptide comprises one or more sequence as disclosed in Table 7 of International Publication Nos. WO 2013/043729 and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 20150132330 which are incorporated herein by reference in their entirety.
  • HAl N-terminal long stem segments share sequence identity between influenza A and influenza B and additionally across influenza A subtypes.
  • HAl C-terminal long stem segments also share sequence identity between influenza A and influenza B and additionally across influenza A subtypes.
  • HA2 domains also share sequence identity between influenza A and influenza B and additionally across influenza A subtypes.
  • influenza hemagglutinin long stem domain polypeptide is a hybrid polypeptide that comprises or consists essentially of segments and/or domains from a plurality of influenza strains or subtypes.
  • influenza strains or subtypes for example, an influenza
  • the HAl N-terminal long stem segment is from influenza A virus while the HAl C-terminal long stem segment is from influenza B virus.
  • HA2 may also be from influenza A virus while the HA1 N-terminal and/or C-terminal long stem segment is from influenza B virus.
  • hemagglutinin HA long stem domain polypeptides of the present invention may be used to form the hemagglutinin HA long stem domain polypeptides of the present invention.
  • a linker covalently connects the HA1 N-terminal long stem segment to the HA1 C-terminal long stem segment.
  • the linker can be any linked deemed suitable by one of skill in the art including, but not limited to, those linkers described herein.
  • influenza hemagglutinin long stem domain polypeptides are capable of forming a three dimensional structure that is similar to the three dimensional structure of the stem domain of a native influenza hemagglutinin. Structural similarity can be evaluated based on any technique deemed suitable by those of skill in the art including, but not limited to, those techniques described herein.
  • GSGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 102
  • a foldon domain can be useful to facilitate trimerization of soluble polypeptides provided herein.
  • Cleavage sites can be used to facilitate cleavage of a portion of a polypeptide, for example cleavage of a purification tag or foldon domain or both.
  • Useful cleavage sites include a thrombin cleavage site, for example one with the sequence LVPRGSP (SEQ ID NO: 103).
  • the cleavage site is a cleavage site recognized by Tobacco Etch Virus (TEV) protease ⁇ e.g., amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) (SEQ ID NO:50).
  • TSV Tobacco Etch Virus
  • influenza hemagglutinin long stem domain polypeptides comprising an elastase cleavage site as described herein.
  • influenza hemagglutinin long stem domain polypeptides that are predicted to be resistant to protease cleavage at the junction between HAl and HA2.
  • Arg-Gly sequence spanning HAl and HA2 is a recognition site for trypsin and is typically cleaved for
  • influenza hemagglutinin activation Since the stem domain polypeptides described herein need not be activated, provided herein are influenza hemagglutinin long stem domain polypeptides that are predicted to be resistant to protease cleavage. In certain embodiments, provided is any influenza hemagglutinin long stem domain polypeptide described herein wherein the protease site spanning HAl and HA2 is mutated to a sequence that is resistant to protease cleavage. In certain embodiments, provided is any influenza hemagglutinin long stem domain polypeptide described herein wherein the C-terminal residue of the HAl C-terminal long stem segment is any residue other than Lys or Arg.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment in binding association with an HA2 stem domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment, in turn covalently linked to an HA2 stem domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment in binding association with an HA2 stem domain that is covalently linked to an HA2 luminal domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of a signal peptide covalently linked to an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain.
  • the linker is a globular head, or a fragment thereof, from an influenza virus heterologous to the influenza stem domain.
  • the linker is a globular head, or a fragment thereof, from an influenza virus heterologous to the stem domain of the HA2 subunit of the hemagglutinin.
  • the linker is a globular head, or a fragment thereof, from an influenza virus heterologous to the stem domain of the HA1 and/or HA2 subunit of the hemagglutinin.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment in binding association with an HA2 stem domain that is covalently linked to, in sequence, a cleavage site, a
  • influenza hemagglutinin long stem domain polypeptides consisting of an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to, in sequence, a cleavage site, a trimerization domain and a purification tag.
  • influenza hemagglutinin long stem domain polypeptides consisting of a signal peptide covalently linked to an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to, in sequence, a cleavage site, a trimerization domain and a purification tag.
  • the protease cleavage site is a thrombin cleavage site.
  • the cleavage site has the amino acid sequence LVPRGSP (SEQ ID NO: 103).
  • the cleavage site is a cleavage site recognized by Tobacco Etch Virus (TEV) protease (e.g., amino acid sequence Glu-Asn-Leu-Tyr- Phe-Gln-(Gly/Ser) (SEQ ID NO: 50).
  • TSV Tobacco Etch Virus
  • the trimerization domain is a foldon domain.
  • the trimerization domain comprises a wildtype GCN4pII trimerization heptad repeat or a modified GCN4pII trimerization heptad repeat that allows for the formation of trimeric or tetrameric coiled coils.
  • the purification tag is a His tag, having the sequence, (Hi s)n, wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HA1 N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HA1 C-terminal long stem segment in binding association with an HA2 stem domain that is covalently linked to an HA2 luminal domain that is covalently linked to, in sequence, a cleavage site, a trimerization domain and a purification tag.
  • influenza hemagglutinin long stem domain polypeptides consisting of a signal peptide covalently linked to an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is covalently linked to, in sequence, a cleavage site, a trimerization domain and a purification tag.
  • the protease cleavage site is a thrombin cleavage site.
  • the cleavage site has the amino acid sequence LVPRGSP (SEQ ID NO: 103).
  • the cleavage site is a cleavage site recognized by Tobacco Etch Virus (TEV) protease (e.g., amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) (SEQ ID NO:50).
  • TSV Tobacco Etch Virus
  • the trimerization domain is a foldon domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment in binding association with an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain.
  • polypeptides consisting of a signal peptide covalently linked to an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain that is in turn covalently linked to an HA2 cytoplasmic domain.
  • influenza hemagglutinin long stem domain polypeptides consisting of a signal peptide covalently linked to an HAl N-terminal long stem segment covalently linked to a linker, in turn covalently linked to an HAl C-terminal long stem segment, in turn covalently linked to an HA2 stem domain that is covalently linked to an HA2 luminal domain that is in turn covalently linked to an HA2 transmembrane domain that is in turn covalently linked to an HA2 cytoplasmic domain.
  • influenza virus hemagglutinin core polypeptides are provided herein.
  • influenza virus hemagglutinin core polypeptide is as described in International Publication No. WO 2011/103453 and U.S. Publication No.
  • the core polypeptide comprises one or more relatively conserved antigenic regions of the HA2 hemagglutinin subunit long alpha-helix.
  • the core polypeptide is capable of generating an immune response in a subject that is capable of cross reacting with, and preferably protecting against, a plurality of influenza virus strains from a single subtype, or strains from 2, 3, 4 or more subtypes.
  • the ability of a core polypeptide to generate an immune response that is capable of neutralizing a plurality of influenza virus strains from a single subtype, or strains from 2, 3, 4 or more subtypes can be assessed using methods known to those of skill in the art and described herein (see Sections 5.13 and 6, of International Publication No. WO 2011/103453 and U.S. Publication No. 2013/0209499, which are incorporated herein by reference in their entirety).
  • the core polypeptide is capable of generating an immune response in a subject that is capable of inhibiting or reducing the replication of a plurality of influenza virus strains from a single subtype, or strains from 2, 3, 4 or more subtypes.
  • a core polypeptide to generate an immune response that is capable of inhibiting or reducing the replication of a plurality of influenza virus strains from a single subtype, or strains from 2, 3, 4 or more subtypes can be assessed using methods known to those of skill in the art and described herein (see Sections 5.13 and 6, of International Publication No. WO 2011/103453 and U.S. Publication No. 2013/0209499, which are incorporated herein by reference in their entirety).
  • One of skill in the art can determine whether or not the alpha-helix conformation is maintained using any method known in the art such as, e.g., NMR, X-ray cry stall ographic methods, or secondary structure prediction methods, e.g., circular dichroism.
  • a core polypeptide does not include the amino acid sequence of a full length influenza virus hemagglutinin.
  • a core polypeptide comprises or consists of between 25 to 50, 50 to 55, 50 to 60, 50 to 65, 50 to 70, 50 to 75, 50 to 80, 50 to 85, 50 to 90, 50 to 95, 50 to 100, 100 to 150, 100 to 200, or 100 to 250 amino acids.
  • a core polypeptide comprises or consists of between 50 to 55, 50 to 60, 50 to 65, 50 to 75, 50 to 80, 50 to 85, 50 to 90, 50 to 95, 50 to 100, 75 to 80, 75 to 85, 75 to 90, 75 to 95, or 75 to 100 amino acids
  • a core polypeptide comprises or consists of amino acids 1( ⁇ 5) to 184( ⁇ 5), 16( ⁇ 5) to 184( ⁇ 5), 30( ⁇ 5) to 184( ⁇ 5), 31 ( ⁇ 5) to 184( ⁇ 5), 46( ⁇ 5) to 184( ⁇ 5), 61 ( ⁇ 5) to 184( ⁇ 5), 70( ⁇ 5) to 110( ⁇ 5), 76( ⁇ 5) to 106( ⁇ 5), 76( ⁇ 5) to 130( ⁇ 5) or 76( ⁇ 5) to 184( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system.
  • a core polypeptide comprises or consists of amino acids 1( ⁇ 5) to 184( ⁇ 5), 16( ⁇ 5) to 184( ⁇ 5), 30( ⁇ 5) to 184( ⁇ 5), 31 ( ⁇ 5) to 184( ⁇ 5), 46( ⁇ 5) to 184( ⁇ 5), 61 ( ⁇ 5) to 184( ⁇ 5), 70( ⁇ 5) to 184( ⁇ 5), (70( ⁇ 5) to 110( ⁇ 5), 76( ⁇ 5) to 106( ⁇ 5), 76( ⁇ 5) to 130( ⁇ 5) or 76( ⁇ 5) to 184( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system, wherein the core polypeptide is less than 300, 275, 250, 200, 190, 185, or 180 amino acids in length.
  • a core polypeptide comprises or consists of amino acids 76 to 106 of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system.
  • a core polypeptide comprises or consists of amino acids 70( ⁇ 5) to 125( ⁇ 5), 80( ⁇ 5) to 115( ⁇ 5), 90( ⁇ 5) to 105( ⁇ 5), or 76( ⁇ 5) to 95( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system.
  • a core polypeptide comprises or consists of amino acids 70( ⁇ 5) to 125( ⁇ 5), 80( ⁇ 5) to 115( ⁇ 5), 90( ⁇ 5) to 105( ⁇ 5), or 76( ⁇ 5) to 95( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system, wherein the core polypeptide is less than 300, 275, 250, 200, 190, 185, 180, 175, 150, 145, 130, 130, 125, 100, or 75 amino acids in length.
  • a core polypeptide comprises or consists of amino acids 70( ⁇ 5) to 130( ⁇ 5), 70( ⁇ 5) to 120( ⁇ 5), 70( ⁇ 5) to 110( ⁇ 5), 70( ⁇ 5) to 100( ⁇ 5), or 70( ⁇ 5) to 95( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system.
  • a core polypeptide comprises or consists of amino acids 70( ⁇ 5) to 130( ⁇ 5), 70( ⁇ 5) to 120( ⁇ 5), 70( ⁇ 5) to 110( ⁇ 5), 70( ⁇ 5) to 100( ⁇ 5), or 70( ⁇ 5) to 95( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system, wherein the core polypeptide is less than 300, 275, 250, 200, 190, 185, 180, 175, 150, 145, 130, 130, 125, 100, or 75 amino acids in length.
  • a core polypeptide comprises or consists of amino acids 70( ⁇ 5) to 130( ⁇ 5), 80( ⁇ 5) to 130( ⁇ 5), 90( ⁇ 5) to 130( ⁇ 5), 100( ⁇ 5) to 130( ⁇ 5), or 110( ⁇ 5) to 130( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system.
  • a core polypeptide comprises or consists of amino acids 70( ⁇ 5) to 130( ⁇ 5), 80( ⁇ 5) to 130( ⁇ 5), 90( ⁇ 5) to 130( ⁇ 5), 100( ⁇ 5) to 130( ⁇ 5), or 110( ⁇ 5) to 130( ⁇ 5) of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system, wherein the core polypeptide is less than 300, 275, 250, 200, 190, 185, 180, 175, 150, 145, 130, 130, 125, 100, or 75 amino acids in length.
  • a core polypeptide comprises or consists of amino acids 1-184, 10( ⁇ 5) to 184, 20( ⁇ 5) to 184, 30( ⁇ 5) to 184, 40( ⁇ 5) to 184, 50( ⁇ 5) to 184, 60( ⁇ 5) to 184, 70( ⁇ 5) to 184 or 80( ⁇ 5) to 184 of a hemagglutinin polypeptide numbered according to the classic H3 subtype numbering system.
  • glycosylation within the stem domain of the polypeptide can hinder or prevent desired immune responses against the conserved antigenic regions found in this domain.
  • an immune response to conserved antigenic regions within the stem domain of the influenza virus HA polypeptide provided herein can be increased by modifying one or more glycosylation sites within the stem domain in a manner that disrupts the glycosylation (i.e. the attachment of a glycan) at the sites.
  • masking of the immunodominant antigenic regions of the HA globular head domain by the addition of one or more non-naturally occurring glycosylation sites in these immunodominant regions can also increase the immunogenicity of conserved subimmunodominant antigenic regions within the stem domain. See Fig. 19C of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety.
  • the flu hemagglutinin (HA) polypeptides comprising one or more modified glycosylation sites and/or one or more non-naturally occurring glycosylation sites can be used in accordance with the methods of vaccination described herein, i.e., such mutant HA polypeptides can be administered to a subject so as to elicit influenza virus stalk/stem domain-specific antibodies in the subject.
  • HA hemagglutinin
  • subjects e.g., mice
  • virus e.g., influenza virus
  • the ability of such mutant HA polypeptides or viruses expressing such mutant HA polypeptides to elicit the production stem/stalk domain specific antibodies can be assessed and compared to the ability of counterpart wild-type HA or wild-type viruses to elicit the production stem/stalk domain specific antibodies in the subject.
  • the flu hemagglutinin (HA) polypeptide provided herein comprises an HA stem domain comprising at least one modified glycosylation site, wherein the modified glycosylation site comprises a modification of a naturally occurring glycosylation site that disrupts the ability of a glycan to attach to the modified glycosylation site.
  • the flu hemagglutinin (HA) polypeptide provided herein comprises an HA stem domain comprising at least one modified glycosylation site as provided in Section 5.4.1 of International Publication No. WO 2013/043729 and U. S. Application No. 14/345,816, which published as U. S. Patent Publication No. 20150132330 which are incorporated herein by reference in their entirety.
  • glycosylation sites within the stem domain in a manner that disrupts the glycosylation (i.e. the attachment of a glycan) at the sites.
  • Modified glycosylation sites in which a naturally occurring glycosylation site is modified in a manner that disrupts the ability of a glycan to attach to the modified glycosylation site can be made by any technique apparent to one of skill in the art, including the methods described herein, including, for example, the site directed mutagenesis techniques discussed in Example 5 of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety.
  • Modified glycosylation sites include, but are not limited to, N-linked and O- linked glycosylation sites.
  • the modified glycosylation site is an N- linked glycosylation site.
  • the modified glycosylation site is an O-linked glycosylation site.
  • the modified glycosylation site is a modified N-linked glycosylation site having the amino acid motif Asn-Xaa-Ser/Thr/Cys, wherein Xaa is any amino acid or, in certain embodiments, wherein Xaa is any amino acid except Pro.
  • the modified glycosylation site can comprise any modification that can disrupt the ability of a glycan to attach to the modified glycosylation site.
  • the modification does not interfere with the proper folding of the flu hemagglutinin (HA) polypeptide and/or the ability of the flu hemagglutinin (HA) polypeptide to elicit an immune response in a subject.
  • the modification comprises a deletion of one or more amino acid residues in a naturally occurring glycosylation site.
  • the modification comprises one or more amino acid substitutions in a naturally occurring
  • the modified glycosylation site comprises one or more amino acid substitutions in a naturally occurring glycosylation site comprising the amino acid sequence Asn-Xaa-Ser/Thr/Cys, wherein Xaa is any amino acid or, in certain embodiments, wherein Xaa is any amino acid except Pro, and wherein the modification disrupts the ability of a glycan to attach to the modified glycosylation site.
  • the modified glycosylation site can comprise any amino acid substitution know to one of skill in art that can disrupt the ability of a glycan to attach to the modified glycosylation site.
  • the one or more amino acid substitutions does not interfere with the ability of the flu hemagglutinin (HA) polypeptide to fold properly or elicit an immune response in a subject.
  • the one or more amino acids of a naturally occurring glycosylation site is substituted for an Asn (N), Ser(s), Thr (T) or Asp (D) amino acid residue.
  • Exemplary amino acid substitutions include, but are not limited to, substitution of an Asn (N) for a Lys (K) amino acid residue; substitution of a Ser(s) for an Asn (N) residue; and substitution of a Thr (T) for an Asp (D) residue.
  • the modified glycosylation site comprises a substitution of an Asn (N) residue of a naturally occurring glycosylation site for a Lys (K) residue. In other embodiments, the modified glycosylation site comprises a substitution of a Ser(s) residue of a naturally occurring glycosylation site for an Asn (N) amino acid residue. In yet other embodiments, the modified glycosylation site comprises a substitution of a Thr (T) residue of a naturally occurring glycosylation site for an Asp (D) amino acid residue.
  • hemagglutinins HI, H2, H5, H6, H8, H9, HI 1, H12, H13, and H16
  • amino acid positions 20-22 missing in H9, 21-23, 33-35 (missing in H8, H9, H12, H13, H16), 46-48 (missing in HI, H2, H5, H6, H8, H9, HI 1, H12), 289-291 (missing in H6, HI 1, H13, H16), 290-292 (missing in HI, H2, H5, H8, H9, H12), 296-298 (missing in HI, H2, H5, HI 1, H13, H16) and 481-483, wherein the amino acid positions are
  • the flu hemagglutinin polypeptide comprising a HA stem domain comprising at least one modified glycosylation site can be any flu hemagglutinin (HA) polypeptide comprising an HA stem domain described herein, including, but not limited to, a chimeric influenza virus hemagglutinin polypeptide, a non-chimeric influenza virus hemagglutinin polypeptide (i.e., an influenza virus hemagglutinin polypeptide comprising an HA stem domain and an HA head domain from the same subtype or strain), and an influenza virus hemagglutinin stem domain polypeptide.
  • a chimeric influenza virus hemagglutinin polypeptide a non-chimeric influenza virus hemagglutinin polypeptide (i.e., an influenza virus hemagglutinin polypeptide comprising an HA stem domain and an HA head domain from the same subtype or strain)
  • an influenza virus hemagglutinin stem domain polypeptide
  • the flu hemagglutinin (HA) polypeptide is a chimeric influenza virus hemagglutinin polypeptide.
  • the chimeric influenza virus hemagglutinin (HA) polypeptide comprises an HA stem domain and an HA globular head domain, wherein the HA globular head domain is heterologous to the HA stem domain, and wherein the HA stem domain comprises at least one modified glycosylation site, wherein the modified glycosylation site comprises a modification of a naturally occurring glycosylation site that disrupts the ability of a glycan to attach to the modified glycosylation site.
  • the modification comprises one or more amino acid substitutions in a naturally occurring glycosylation site having the amino acid sequence Asn-Xaa-Ser/Thr/Cys, wherein Xaa is any amino acid or, in certain embodiments, wherein Xaa is any amino acid except Pro.
  • the non-chimeric influenza virus hemagglutinin polypeptide comprises an HA stem domain and HA globular head domain from the same influenza virus subtype.
  • the influenza virus subtype is an HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18 subtype.
  • the non- chimeric influenza virus hemagglutinin polypeptide comprises an HA stem domain and HA globular head domain from the same influenza virus strain.
  • influenza virus strain is A/Netherlands/602/2009.
  • the flu hemagglutinin (HA) polypeptide is an influenza virus hemagglutinin stem domain polypeptide.
  • influenza virus hemagglutinin stem domain polypeptides are disclosed in Section 5.3, supra.
  • Non-naturally occurring glycosylation sites can be added to the HA globular head domain of the flu hemagglutinin (HA) polypeptide described herein using any known technique known to one of skill in the art, including, for example, the site directed mutagenesis techniques described in Example 5 of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety.
  • the non-naturally occurring glycosylation site does not interfere with the proper folding of the flu hemagglutinin (HA) polypeptide and/or interfere with the ability of the stem domain of the flu hemagglutinin (HA) polypeptide from eliciting an immune response (e.g., an antibody response) in a subject.
  • the non-naturally occurring glycosylation sites can be added to an HA globular head domain based on the head domain of an influenza A
  • the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with two non-naturally occurring glycosylation sites. In specific embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with three non-naturally occurring glycosylation sites. In other embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with four non-naturally occurring glycosylation sites. In certain embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with five non-naturally occurring glycosylation sites.
  • the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with ten non-naturally occurring glycosylation sites. In other embodiments, the flu hemagglutinin (HA) polypeptide an HA globular head domain with eleven non-naturally occurring glycosylation sites. In other embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with twelve non-naturally occurring glycosylation sites. In certain embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with thirteen non-naturally occurring glycosylation sites.
  • the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with fourteen non-naturally occurring glycosylation sites. In other embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with fifteen non- naturally occurring glycosylation sites. In other embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with sixteen non-naturally occurring glycosylation sites. In certain embodiments, the flu hemagglutinin (HA) polypeptide comprises an HA globular head domain with seventeen non-naturally occurring glycosylation sites.
  • glycosylation site is at amino acid position 129-131, according to H3 numbering. In other embodiments, the non-naturally occurring glycosylation sites are at amino acid positions 129- 131 and 158-160, according to H3 numbering. In some embodiments, the non-naturally occurring glycosylation sites are at amino acid positions 59-61, 129-131 and 165-167, according to H3 numbering. In some embodiments, the non-naturally occurring glycosylation sites are at amino acid positions 59-61, 129-131, 158-160 and 165-167, according to H3 numbering.
  • the non-naturally occurring glycosylation sites are at amino acid positions 81-83, 129-131, 158-160, 165-167, 170-172, 187-189 and 208-210, according to H3 numbering. In other embodiments, the non-naturally occurring glycosylation sites are at amino acid positions 81-83, 129-131, 158-160, 170-172, 187-189 and 208-210, according to H3 numbering. In still other embodiments, the non-naturally occurring glycosylation sites are at amino acid positions 129-131, 158-160, 165-167, 170-172, 187-189 and 208-210, according to H3 numbering.
  • the non-naturally occurring glycosylation site is located in an antigenic region in the globular head domain, thereby shielding the antigenic region from eliciting an immune response.
  • Exemplary antigenic regions in the globular domain include, but are not limited to the Sa, Sb, Ca and Cb antigenic site (Fig. 21 A of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety) in the HI subtype and the A, B, C, D antigenic regions in the H3 subtype.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sa antigenic region of an HI subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sb antigenic region of an HI subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Ca antigenic region of an HI subtype globular head domain. In yet other embodiments, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Cb antigenic region of an HI subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non- naturally occurring glycosylation site located in the Sa and Sb antigenic regions of an HI subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sa and Ca antigenic regions of an HI subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sa and Cb antigenic regions of an HI subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sb and Ca antigenic regions of an HI subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sb and Cb antigenic regions of an HI subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non- naturally occurring glycosylation site located in the Ca and Cb antigenic regions of an HI subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sa, Sb, and Ca antigenic regions of an HI subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sb, Ca and Cb antigenic regions of an HI subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the Sa, Sb, Ca and Cb antigenic regions of an HI subtype globular head domain.
  • the non-naturally occurring glycosylation site is in the A antigenic region of an H3 subtype globular head domain. In some embodiments, the non- naturally occurring glycosylation site is in the B antigenic region of an H3 subtype globular head domain. In some embodiments, the non-naturally occurring glycosylation site is in the C antigenic region of an H3 subtype globular head domain. In some embodiments, the non-natural occurring glycosylation site is in the D antigenic region of an H3 subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non- naturally occurring glycosylation site located in the A and B antigenic regions of an H3 subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the A and C antigenic regions of an H3 subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the A and D antigenic regions of an H3 subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the B and C antigenic regions of an H3 subtype globular head domain. In another
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the B and D antigenic regions of an H3 subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non- naturally occurring glycosylation site located in the C and D antigenic regions of an H3 subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the A, B, and C antigenic regions of an H3 subtype globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the B, C, and D antigenic regions of an H3 subtype globular head domain. In another embodiment, the flu hemagglutinin (HA) polypeptide comprises a non-naturally occurring glycosylation site located in the A, B, C, and D antigenic regions of an H3 subtype globular head domain.
  • a flu hemagglutinin (HA) polypeptide comprises one or more non-naturally occurring glycosylation sites in one or more antigenic regions of an HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16 or H17 globular head domain.
  • the flu hemagglutinin (HA) polypeptide comprising an
  • HA globular head domain with one or more non-naturally occurring glycosylation sites is a chimeric influenza virus hemagglutinin polypeptide.
  • hemagglutinin (HA) polypeptide comprising an HA globular head domain with one or more non- naturally occurring glycosylation sites is a non-chimeric influenza virus hemagglutinin polypeptide.

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