WO2019032463A1

WO2019032463A1 - Immunoginic compositions comprising chimeric influenza virus hemagglutinin polypeptides and as01 and uses thereof

Info

Publication number: WO2019032463A1
Application number: PCT/US2018/045399
Authority: WO
Inventors: Peter Palese; Florian KRAMMER; Adolfo Garcia-Sastre; Raffael NACHBAGAUER; Corey Patrick MALLETT; Bruce Innis; Emmanuel Jules Hanon
Original assignee: Icahn School Of Medicine At Mount Sinai; Glaxosmithline Biologicals S.A.
Priority date: 2017-08-07
Filing date: 2018-08-06
Publication date: 2019-02-14

Abstract

Provided herein are immunization regimens for inducing an immune response (e.g., an antibody response) against influenza virus. In specific aspects, the immunization regimens involve the administration of a chimeric hemagglutinin (HA) and a liposomal adjuvant (e.g., ASOl) to a subject. Also provided herein are immunogenic/vaccine compositions for use in methods of immunizing or immunization regimens for human subjects against influenza virus.

Description

IMMUNOGENIC COMPOSITIONS COMPRISING CHIMERIC INFLUENZA VIRUS HEMAGGLUTININ POLYPEPTIDES AND AS01 AND USES THEREOF

[0001] This application claims the benefit of U. S. Provisional Application No. 62/542, 143, filed August 7, 2017, which is incorporated by reference herein in its entirety.

[0002] This invention was made with government support under P01 AI097092 and

HHSN272201400008C awarded by NIH. The government has certain rights in the invention.

[0003] This application incorporates by reference a Sequence Listing submitted with this application as text file entitled "Seqlisting_6923_274_228_ST25.txt" created on August 5, 2018 and having a size of 301 kilobytes.

1. INTRODUCTION

[0004] Provided herein are compositions comprising a chimeric influenza virus

hemagglutinin (HA) polypeptide and a liposomal adjuvant (e.g., AS01). In specific aspects, provided herein are compositions comprising an inactivated influenza virus and a liposomal adjuvant (e.g., AS01), wherein the influenza virus contains a chimeric HA polypeptide. Also provided herein are immunization/vaccination regimens for inducing an immune response (e.g., an antibody response) against influenza virus involving the administration of such compositions to a human subject.

2. BACKGROUND

[0005] Influenza viruses are enveloped RNA viruses that belong to the family of

Orthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Viruses and Their

Replication, 5th ed. Fields' Virology, edited by B.N. Fields, D.M. Knipe and P.M. Howley. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, USA, pi 647- 1689). The natural host of 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). For example, the H5N1 avian 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). The occurrence of influenza virus infections in animals could potentially give rise to human pandemic influenza strains.

[0006] 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. In a normal season, 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). In the United States, influenza viruses infect an estimated 10-15% of the population (Glezen and Couch RB (1978) Interpandemic influenza in the Houston area, 1974-76. N Engl J Med 298: 587-592; Fox et al. (1982) Influenza virus infections in Seattle families, 1975-1979. II. Pattern of infection in invaded households and relation of age and prior antibody to occurrence of infection and related illness. Am J Epidemiol 116: 228-242) and are associated with approximately 30,000 deaths each year (Thompson WW et al. (2003) Mortality Associated with Influenza and Respiratory Syncytial Virus in the United States. JAMA 289: 179-186; Belshe (2007) Translational research on vaccines: influenza as an example. Clin Pharmacol Ther 82: 745-749).

[0007] In addition to annual epidemics, influenza viruses are the cause of infrequent pandemics. For example, 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. A stark example is the 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). Since the emergence of the highly pathogenic avian H5N1 influenza virus in the late 1990s (Claas et al. (1998) Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351 : 472-7), there have been concerns that it may be the next pandemic virus. Further, H7 and H9 strains are candidates for new pandemics since these strains infect humans on occasion.

[0008] An effective way to protect against influenza virus infection is through vaccination; however, current vaccination approaches rely on achieving a good match between circulating strains and the isolates included in the vaccine. Such a match is often difficult to attain due to a combination of factors. First, 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.

[0009] The possibility of a novel subtype of influenza A virus entering the human population also presents a significant challenge to current vaccination strategies. Since it is impossible to predict what subtype and strain of influenza virus will cause the next pandemic, current, strain- specific approaches cannot be used to prepare a pandemic influenza vaccine in advance of a pandemic. Thus, there is a need for vaccines that cross-protect subjects against different strains and/or subtypes of influenza virus.

3. SUMMARY

[0010] The invention is based, in part, on the discovery that when ASOl was used in combination with an influenza virus chimeric HA polypeptide in a split virus vaccine administered intramuscularly, a higher influenza specific-T cell response in the lungs was detected relative to the influenza virus-specific T cell response detected when the chimeric HA polypeptide was administered intramuscularly in a split virus vaccine without adjuvant or with AS03. See FIG. 4, infra - see, in particular, the results obtained in mice vaccinated with QIV- IIV-IIV plus ASOl, AS03 or unadjuvanted. In addition, titers for mouse IgG2a (which is similar to human IgGl and human IgG3 in their ability to elicit Fc-mediated effector functions) were higher when the ASOl adjuvant was used in combination with the chimeric HA polypeptide in a split virus vaccine relative to when the chimeric HA polypeptide was administered in a split virus vaccine without adjuvant or with AS03. See FIG. 41, infra. Further, a higher ratio of IgG2a to IgGl was detected in mice intramuscularly administered a chimeric HA polypeptide in a split virus vaccine relative to mice intramuscularly administered the chimeric HA polypeptide in a split virus vaccine without adjuvant or with AS03. See FIG. 41, infra. The IgG2a/IgGl ratio in animals vaccinated with a chimeric HA polypeptide in a split virus vaccine with AS01 are consistent with a Thl-focused response, which indicates a better T cell response. The influenza virus-specific T cell response in the lungs in combination with the higher ratio of IgG2a to IgGl indicates that a better T cell response is induced when a liposomal adjuvant such as AS01 is used in combination with a chimeric influenza virus HA polypeptide in a split virus vaccine administered intramuscularly. The induction of a higher T cell response indicates a more robust immune response to the influenza virus and potentially offers additional protection against a subsequent influenza virus infection.

[0011] Provided herein is an immunogenic composition comprising a chimeric influenza virus hemagglutinin (HA) polypeptide and a liposomal adjuvant, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. In certain embodiments, the immunogenic composition is a subunit vaccine.

[0012] Also provided herein is an immunogenic composition comprising an inactivated influenza virus and a liposomal adjuvant, wherein the inactivated influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

[0013] Also provided herein is an immunogenic composition comprising a split influenza virus and a liposomal adjuvant, wherein the split influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

[0014] In specific embodiments, an immunogenic composition described herein comprises a first chimeric HA polypeptide, a second chimeric HA polypeptide and a third chimeric HA polypeptide, wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or HI 5), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of an influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the influenza B virus HA globular head domain, and the HA stem domain of the influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain. In specific embodiments, an immunogenic composition described herein comprises a first chimeric HA polypeptide, a second chimeric HA polypeptide, a third chimeric HA polypeptide and adjuvant (in specific embodiments, a liposomal adjuvant such as described in Section 5.8.5 below or Section 6 below), wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of an influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the influenza B virus HA globular head domain, and the HA stem domain of the influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain. The immunogenic composition may be an inactivated vaccine, such as subunit vaccine, split vaccine or whole inactivated virus vaccine. In particular embodiments, the first and second chimeric HA polypeptides may be ones described in Section 5.1 below (in particular, Section 5.1.1.1 below), Section 6.1 below, Section 6.2 below, Section 6.3 below, or Section 6.4 below and the third chimeric HA polypeptide may be one described in Section 5.1.2 below or Section 6.5 below.

[0015] In a specific embodiment, an immunogenic composition is an immunogenic composition comprising one, two, three or more chimeric influenza virus HA polypeptides described herein (e.g., Section 5.1 below or Section 6 below). In another specific embodiment, an immunogenic composition is an immunogenic composition described herein (e.g., in Seection 5.8 below or Section 6 below). The immunogenic compositions described herein may be used to immunize a subject (e.g., a human subject) against an influenza virus. The immunogenic compositions described herein may prevent or treat, or both, an influenza virus infection or an influenza virus disease.

[0016] In certain embodiments, the influenza virus HA globular head domain in an immunogenic composition provided herein is from one influenza A virus subtype and the HA stem domain polypeptide is from a different influenza A virus subtype. In certain embodiments, the HA stem domain in an immunogenic composition provided herein is from influenza A virus subtype HI . In other embodiments, the HA stem domain is from influenza A virus subtype H3. In certain embodiments, the influenza virus HA globular head domain of an immunogenic composition provided herein is from influenza A virus H8. In other embodiments, the influenza virus HA globular head domain is from influenza A virus H5. In yet other embodiments, the influenza virus HA globular head domain is from influenza A virus subtype H4, H6, H7, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18. In certain embodiments, the influenza virus HA globular head domain is from one influenza virus species and the HA stem domain is from a different influenza virus species. In certain embodiments, the influenza virus HA globular head domain is from an influenza A virus and the HA stem domain polypeptide is from an influenza B virus lineage. [0017] In certain embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide in an immunogenic composition provided herein is from one influenza A virus subtype and the HA stem domain polypeptide is from a different influenza A virus subtype. In certain embodiments, the HA stem domain of a chimeric HA polypeptide in an immunogenic composition provided herein is from influenza A virus subtype HI . In other embodiments, the HA stem domain of a chimeric HA polypeptide is from influenza A virus subtype H3. In certain embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide provided herein is from influenza A virus H8. In other embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide is from influenza A virus H5. In yet other embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide is from influenza A virus subtype H4, H6, H7, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or HI 8. In certain embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide is from one influenza virus species and the HA stem domain is from a different influenza virus species. In certain embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide is from an influenza A virus and the HA stem domain polypeptide is from an influenza B virus lineage.

[0018] In certain embodiments, the influenza virus HA globular head domain in an immunogenic composition provided herein consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering. In specific embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide in an immunogenic composition provided herein consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

[0019] In certain embodiments, the HA stem domain polypeptide in an immunogenic composition provided herein 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 HAlN-term through Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering. In specific embodiments, the HA stem domain polypeptide in an immunogenic composition provided herein comprises an HAl N-terminal stem segment, an HAl C-terminal stem segment, and an HA2 stem domain, wherein the HAl N-terminal stem segment consists of amino acid residues HAlN-term through Ap of an influenza A virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

[0020] In certain embodiments, a chimeric HA polypeptide in an immunogenic composition provided herein comprises an influenza A virus HA stem domain and an influenza A virus HA globular head domain, wherein the HA stem domain 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 HAlN-term through Ap of an influenza A virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq. In specific embodiments, a chimeric HA polypeptide in an immunogenic composition provided herein comprises an influenza A virus HA stem domain and an influenza A virus HA globular head domain, wherein the HA stem domain comprises an HAl N-terminal stem segment, an HAl C- terminal stem segment and an HA2 stem domain, wherein the HAl N-terminal stem segment consists of amino acid residues HAlN-term through Ap of an influenza A virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq.

[0021] In certain embodiments, the HA stem domain polypeptide in an immunogenic composition provided herein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering.

[0022] In specific embodiments, the HA stem domain polypeptide of a chimeric HA polypeptide in an immunogenic composition provided herein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering.

[0023] In certain embodiments, a chimeric HA polypeptide in an immunogenic composition provided herein comprises an HA stem domain and an HA globular head domain, wherein the HA stem domain 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C- terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HA0 protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

[0024] In certain embodiments, the HA stem domain polypeptide in an immunogenic composition provided herein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HA0 protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N-terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq in the HAl C-terminal stem segment is substituted with a Cys.

[0025] In specific embodiments, the HA stem domain polypeptide of a chimeric HA polypeptide in an immunogenic composition provided herein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N-terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq in the HAl C-terminal stem segment is substituted with a Cys.

[0026] In certain embodiments, a chimeric HA polypeptide in an immunogenic composition provided herein comprises an HA stem domain and an HA globular head domain, wherein the HA stem domain 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C- terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N- terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to

B/Yamagata/16/88 numbering, wherein Dq in the HAl C-terminal stem segment is substituted with a Cys, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

[0027] In certain embodiments, the influenza virus HA globular head domain in an immunogenic composition provided herein consists of the amino acid residues intervening Dp and Dq, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HA1 domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering.

[0028] In specific embodiments, the influenza virus HA globular head domain of a chimeric HA polypeptide in an immunogenic composition provided herein consists of the amino acid residues intervening Dp and Dq, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering.

[0029] In certain embodiments, the influenza virus HA stem domain in the immunogenic composition further comprises an influenza virus HA2 domain lacking the cytoplasmic domain and transmembrane domain. In specific embodiments, the influenza virus HA stem domain of a chimeric HA polypeptide of an immunogenic composition described herein further comprises an influenza virus HA2 domain lacking the cytoplasmic domain and transmembrane domain.

[0030] In certain embodiments, a chimeric HA polypeptide in an immunogenic composition described herein is from an influenza B virus HA, and wherein the HA globular head domain is the globular head domain derived from the influenza B virus HA and comprises one, two, three or all of the following: (a) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (b) 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (c) 2, 3, 4, 5 or more amino acid substitutions within the 160 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; and (d) 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In certain embodiments, the chimeric influenza virus HA polypeptide further comprises the transmembrane domain and cytoplasmic tail domain of the influenza B virus HA. In certain embodiments, the influenza B virus is of the Yamagata lineage or of the Victoria lineage. In certain embodiments, the influenza B virus is influenza B/Yamagata/16/88. In certain embodiments, the influenza A virus is an influenza A virus of an H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18 subtype. In certain embodiments, the influenza A virus is an H5 HA subtype. In certain embodiments in which the influenza A virus is an H5 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues FIP and KIQLSTKNVINAEHAPGGPYRL (SEQ ID NO: 109); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PYQGKSS (SEQ ID NO: 124); (c) the acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KKNSTY (SEQ ID NO: 111); and/or (d) the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NDAAMQT (SEQ ID NO: 113). In certain embodiments, the influenza A virus is an H8 HA subtype. In certain embodiments in which the influenza A virus is an H8 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues HIP and

RIRLSTYNVINAETAPGGPYRL (SEQ ID NO: 125); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NASTGGQS (SEQ ID NO: 126); (c) the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KKKADTY (SEQ ID NO: 127); and/or (d) the amino acid residues

NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues ADAKMQT (SEQ ID NO: 128). In certain embodiments, the influenza A virus is an HI 1 HA subtype. In certain embodiments in which the influenza A virus is an HI 1 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LIP and KIEL S T SN VIN AE V AP GGP YRL (SEQ ID NO: 129); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PFGSSNS (SEQ ID NO: 130); (c) the amino acid residues RDNKTA (SEQ ID NO: 1 10) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues HQSGTY (SEQ ID NO: 131); and/or (d) the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus

B/Yamagata/16/88 are substituted with amino acid residues TTLKMHQ (SEQ ID NO: 132). In certain embodiments, the influenza A virus is an H12 HA subtype. In certain embodiments in which the influenza A virus is an H12 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and

NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues YIP and

RIKL STFNVINAET APGGP YRL (SEQ ID NO: 136); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NNTSNQGS (SEQ ID NO: 137); (c) the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LKSGQF (SEQ ID NO: 138); and/or (d) the amino acid residues

NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PTSDMQI (SEQ ID NO: 139). In certain embodiments, the influenza A virus is an HI 3 HA subtype. In certain embodiments in which the influenza A virus is an HI 3 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/ 16/88 are substituted with amino acid residues NIP and RIEL S THN VIN AE V AP GGP YRL (SEQ ID NO: 168); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/ 16/88 are substituted with amino acid residues PDKGASS (SEQ ID NO: 133); (c) the amino acid residues RDNKTA (SEQ ID NO: 1 10) in the 160 loop of influenza B virus B/Yamagata/ 16/88 are substituted with amino acid residues KRGNQY (SEQ ID NO: 134); and/or (d) the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus

B/Yamagata/ 16/88 are substituted with amino acid residues VSTNMAK (SEQ ID NO: 135).

[0031] Also provided herein are immunogenic compositions for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), wherein the method comprises administering the immunogenic composition to the subject.

[0032] Also provided herein is a method for immunizing against influenza virus in a subject (e.g., a human subject), comprising administering to the subject an immunogenic composition described herein (e.g., an immunogenic composition described above or Section 5.8 below or Section 6 below).

[0033] Also provided herein is a method for immunizing against an influenza virus in a subject (e.g., a human subject), comprising: (a) administering to the subject an immunogenic composition comprising a chimeric influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. See Section 5.1 below and Section 6 below for examples of chimeric influenza virus HA polypeptides that may be present in an immunogenic composition.

[0034] Also provided herein is a method for immunizing against an influenza virus in a subject (e.g., a human subject), comprising: (a) administering to the subject an immunogenic composition comprising an inactivated influenza virus, wherein the inactivated influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. See Section 5.1 below and Section 6 below for examples of chimeric influenza virus HA polypeptides that may be present in an immunogenic composition.

[0035] Also provided herein is a method of immunizing against influenza virus in a subject (e.g., a human subject), comprising: (a) administering to the subject an immunogenic composition comprising a split influenza virus and a liposomal adjuvant, wherein the split influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. See Section 5.1 below and Section 6 below for examples of chimeric influenza virus HA polypeptides that may be present in an immunogenic composition.

[0036] Also provided herein is an immunogenic composition for use in a method for immunizing against an influenza virus in a subject (e.g., a human subject), the method comprising: (a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises a chimeric influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. In a specific embodiment^ he immunogenic composition is a subunit vaccine. See Section 5.1 below and Section 6 below for examples of chimeric influenza virus HA polypeptides that may be present in an immunogenic composition.

[0037] Also provided herein is an immunogenic composition for use in a method for immunizing against an influenza virus in a subject (e.g., a human subject), the method comprising: (a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises an inactivated influenza virus, wherein the inactivated influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. See Section 5.1 below and Section 6 below for examples of chimeric influenza virus HA polypeptides that may be present in an immunogenic composition.

[0038] Also provided herein is an immunogenic composition for use in a method of immunizing against influenza virus in a subject (e.g., a human subject), the method comprising: (a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises a split influenza virus and a liposomal adjuvant, wherein the split influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. See Section 5.1 below and Section 6 below for examples of chimeric influenza virus HA polypeptides that may be present in an immunogenic composition.

[0039] Also provided herein is a method for immunizing against influenza virus in a human subject, the method comprising: (a) administering to the subject a first immunogenic

composition comprising a first chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a first HA subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus of a second HA subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and (b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second

immunogenic composition comprises a second chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a third HA subtype, and wherein the first, second and third HA subtypes are different subtypes. In a specific embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H5 subtype and the third HA subtype is an H8. In another specific embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H8 subtype and the third HA subtype is an H5. In some embodiments, the first immunogenic, second immunogenic composition or both are a subunit vaccine. In certain embodiments, the first immunogenic, second immunogenic composition or both are a split vaccine. In some embodiments, the first immunogenic, second immunogenic composition or both are an inactivated influenza virus vaccine. In certain embodiments, the method further comprises administering a third immunogenic composition comprising a third chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the third chimeric influenza virus HA polypeptide comprises a third influenza virus HA globular head domain and the first influenza virus stem domain, wherein the third influenza virus HA globular head domain comprises the HA globular head domain of an influenza A virus of a fourth HA subtype, and wherein the first, second, third and fourth HA subytpes are different subtypes. In some embodiments, the fourth immunogenic composition is a split vaccine, an inactivated influenza virus vaccine, or a subunit vaccine. See Section 5.1 below (in particular, Section 5.1.1 below) and Section 6 below for examples of chimeric HA polypeptides that may be present in an immunogenic composition.

[0040] Also provided herein is a method for immunizing against influenza virus in a human subject, the method comprising administering to the subject a second immunogenic composition a certain time after the administration of a first immunogenic composition, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a third HA subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus of a second HA subtype, wherein the first immunogenic composition comprises a first chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the first chimeric influenza virus HA

polypeptide comprises a first influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a first HA subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and wherein the first, second and third HA subtypes are different subtypes. In a specific embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H5 subtype and the third HA subtype is an H8. In another specific embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H8 subtype and the third HA subtype is an H5. In some embodiments, the first immunogenic, second immunogenic composition or both are a subunit vaccine. In certain embodiments, the first immunogenic, second immunogenic composition or both are a split vaccine. In some embodiments, the first immunogenic, second immunogenic composition or both are an inactivated influenza virus vaccine. In certain embodiments, the method further comprises administering a third immunogenic composition comprising a third chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the third chimeric influenza virus HA polypeptide comprises a third influenza virus HA globular head domain and the first influenza virus stem domain, wherein the third influenza virus HA globular head domain comprises the HA globular head domain of an influenza A virus of a fourth HA subtype, and wherein the first, second, third and fourth HA subytpes are different subtypes. In a specific embodiment, the fourth HA subtype is an HI 1 subtype. In some embodiments, the fourth immunogenic composition is a split vaccine, an inactivated influenza virus vaccine, or a subunit vaccine. See Section 5.1 below (in particular, Section 5.1.1 below) and Section 6 below for examples of chimeric HA polypeptides that may be present in an immunogenic composition.

[0041] Also provided herein is a first immunogenic composition for use in a method for immunizing against influenza virus in a human subject, the method comprising: (a)

administering to the subject the first immunogenic composition, wherein the first immunogenic composition comprises a first chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a first HA subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus of a second HA subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and (b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a third HA subtype, and wherein the first, second and third HA subtypes are different subtypes. In a specific embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H5 subtype and the third HA subtype is an H8. In another specific embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H8 subtype and the third HA subtype is an H5. In some embodiments, the first immunogenic, second immunogenic composition or both are a subunit vaccine. In certain embodiments, the first immunogenic, second immunogenic composition or both are a split vaccine. In some embodiments, the first immunogenic, second immunogenic composition or both are an inactivated influenza virus vaccine. In certain embodiments, the method further comprises administering a third

immunogenic composition comprising a third chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the third chimeric influenza virus HA polypeptide comprises a third influenza virus HA globular head domain and the first influenza virus stem domain, wherein the third influenza virus HA globular head domain comprises the HA globular head domain of an influenza A virus of a fourth HA subtype, and wherein the first, second, third and fourth HA subytpes are different subtypes. In a specific embodiment, the fourth HA subtype is an HI 1 subtype. In some embodiments, the fourth immunogenic composition is a split vaccine, an inactivated influenza virus vaccine, or a subunit vaccine. See Section 5.1 below (in particular, Section 5.1.1 below) and Section 6 below for examples of chimeric HA polypeptides that may be present in an immunogenic composition. [0042] Also provided herein is a second immunogenic composition for use in a method for immunizing against influenza virus in a human subject, the method comprising administering to the subject the second immunogenic composition a certain time after the administration of a first immunogenic composition, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a third HA subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus of a second HA subtype, wherein the first immunogenic composition comprises a first chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus of a first HA subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and wherein the first, second and third HA subtypes are different subtypes. In a specific embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H5 subtype and the third HA subtype is an H8. In another specific

embodiment, the second HA subtype is an HI subtype, the first HA subtype is an H8 subtype and the third HA subtype is an H5. In some embodiments, the first immunogenic, second immunogenic composition or both are a subunit vaccine. In certain embodiments, the first immunogenic, second immunogenic composition or both are a split vaccine. In some embodiments, the first immunogenic, second immunogenic composition or both are an inactivated influenza virus vaccine. In certain embodiments, the method further comprises administering a third immunogenic composition comprising a third chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the third chimeric influenza virus HA polypeptide comprises a third influenza virus HA globular head domain and the first influenza virus stem domain, wherein the third influenza virus HA globular head domain comprises the HA globular head domain of an influenza A virus of a fourth HA subtype, and wherein the first, second, third and fourth HA subytpes are different subtypes. In a specific embodiment, the fourth HA subtype is an HI 1 subtype. In some embodiments, the fourth immunogenic composition is a split vaccine, an inactivated influenza vims vaccine, or a subunit vaccine. See Section 5.1 below (in particular, Section 5.1.1 below) and Section 6 below for examples of chimeric HA polypeptides that may be present in an immunogenic composition.

[0043] In specific embodiments, a method for immunizing a subject against influenza virus comprising: (A) administering to the subject a first immunogenic composition comprising a first chimeric HA polypeptide, a second chimeric HA polypeptide and a third chimeric HA polypeptide, wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or HI 5), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises the a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of a first influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the first influenza B virus HA globular head domain, and the third HA stem domain comprises the HA stem domain of the first influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain; (B) administering to the subject a second immunogenic composition comprising a fourth chimeric HA polypeptide, a fifth chimeric HA polypeptide and a sixth chimeric HA polypeptide, wherein (i) the fourth chimeric HA polypeptide comprises a fourth HA globular head domain of a fifth influenza A virus of a fifth HA subtype and a fourth HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the fourth HA globular head domain is heterologous to the fourth HA stem domain, (ii) the fifth chimeric HA polypeptide comprises a fifth HA globular head domain of a sixth influenza A virus of a sixth HA subtype and a fifth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the fifth HA globular head domain is heterologous to the fifth HA stem domain, wherein the second, fourth, fifth and sixth HA subtypes are different from each other, and (iii) the sixth chimeric HA polypeptide comprises a sixth HA globular head domain and a sixth HA stem domain, wherein the sixth HA globular head domain comprises the HA globular head domain of a second influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the second influenza B virus HA globular head domain, and the sixth HA stem domain comprises the HA stem domain of the second influenza B virus, wherein the sixth HA globular head domain is heterologous to the sixth HA stem domain; and (C) administering to the subject a third immunogenic composition comprising a seventh chimeric HA polypeptide, an eighth chimeric HA polypeptide and a ninth chimeric HA polypeptide, wherein (i) the seventh chimeric HA polypeptide comprises a seventh HA globular head domain of a seventh influenza A virus of a seventh HA subtype and a seventh HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the seventh HA globular head domain is heterologous to the seventh HA stem domain, (ii) the eighth chimeric HA polypeptide comprises an eighth HA globular head domain of an eighth influenza A virus of an eighth HA subtype and an eighth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the eighth HA globular head domain is heterologous to the eighth HA stem domain, wherein the second, fourth, seventh and eighth HA subtypes are different from each other, and (iii) the ninth chimeric HA polypeptide comprises a ninth HA globular head domain and a ninth HA stem domain, wherein the ninth HA globular head domain comprises the HA globular head domain of a third influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the third influenza B virus HA globular head domain, and the ninth HA stem domain comprises the HA stem domain of the third influenza B virus, wherein the ninth HA globular head domain is heterologous to the ninth HA stem domain. In a specific embodiment, the first, second, third, fourth, fifth, sixth, seventh and eighth HA substypes are different from each other. In another specific embodiment, the first, second and third influenza B viruses are different from each other. In yet other

embodiments, the first, second and third influenza B viruses are the same. The either one, two or all of the first, second and third immunogenic compositions may comprise an adjuvant (e.g., a liposomal adjuvant such as described in Section 5.8.5 below or Section 6 below). The immunogenic composition may be an inactivated vaccine, such as subunit vaccine, split vaccine or whole inactivated virus vaccine. In particular embodiments, the first, second, fourth, fifth, seventh, and eighth chimeric HA polypeptides may be ones described in Section 5.1.1 below (in particular, Section 5.1.1.1 below), Section 6.1 below, Section 6.2 below, Section 6.3 below, or Section 6.4 below and the third, sixth and ninth chimeric HA polypeptide may be one described in Section 5.1.2 below or Section 6.5 below.

[0044] In specific embodiments, a method for immunizing a subject against influenza virus comprising: (A) administering to the subject a first immunogenic composition comprising a first chimeric HA polypeptide, a second chimeric HA polypeptide, a third chimeric HA polypeptide and an adjuvant (in specific embodiments, a liposomal adjuvant such as described in Section 5.8.5 below or Section 6 below), wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises the a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of a first influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the first influenza B virus HA globular head domain, and the third HA stem domain comprises the HA stem domain of the first influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain; (B) administering to the subject a second immunogenic composition comprising a fourth chimeric HA polypeptide, a fifth chimeric HA polypeptide, a sixth chimeric HA polypeptide and an adjuvant (in specific embodiments, a liposomal adjuvant such as described in Section 5.8.5 below or Section 6 below), wherein (i) the fourth chimeric HA polypeptide comprises a fourth HA globular head domain of a fifth influenza A virus of a fifth HA subtype and a fourth HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the fourth HA globular head domain is heterologous to the fourth HA stem domain, (ii) the fifth chimeric HA

polypeptide comprises a fifth HA globular head domain of a sixth influenza A virus of a sixth HA subtype and a fifth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the fifth HA globular head domain is heterologous to the fifth HA stem domain, wherein the second, fourth, fifth and sixth HA subtypes are different from each other, and (iii) the sixth chimeric HA polypeptide comprises a sixth HA globular head domain and a sixth HA stem domain, wherein the sixth HA globular head domain comprises the HA globular head domain of a second influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the second influenza B virus HA globular head domain, and the sixth HA stem domain of the HA stem domain of the second influenza B virus, wherein the sixth HA globular head domain is heterologous to the sixth HA stem domain; and (C) administering to the subject a third immunogenic composition comprising a seventh chimeric HA polypeptide, an eighth chimeric HA polypeptide, a ninth chimeric HA polypeptide and an adjuvant (in specific embodiments, a liposomal adjuvant such as described in Section 5.8.5 below or Section 6 below), wherein (i) the seventh chimeric HA polypeptide comprises a seventh HA globular head domain of a seventh influenza A virus of a seventh HA subtype and a seventh HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the seventh HA globular head domain is heterologous to the seventh HA stem domain, (ii) the eighth chimeric HA polypeptide comprises an eighth HA globular head domain of an eighth influenza A virus of an eighth HA subtype and an eighth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the eighth HA globular head domain is heterologous to the eighth HA stem domain, wherein the second, fourth, seventh and eighth HA subtypes are different from each other, and (iii) the ninth chimeric HA polypeptide comprises a ninth HA globular head domain and a ninth HA stem domain, wherein the ninth HA globular head domain comprises the HA globular head domain of a third influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the third influenza B virus HA globular head domain, and the ninth HA stem domain comprises the HA stem domain of the third influenza B virus, wherein the ninth HA globular head domain is heterologous to the ninth HA stem domain. In a specific embodiment, the first, second, third, fourth, fifth, sixth, seventh and eighth HA substypes are different from each other. In another specific embodiment, the first, second and third influenza B viruses are different from each other. In yet other embodiments, the first, second and third influenza B viruses are the same. The immunogenic composition may be an inactivated vaccine, such as subunit vaccine, split vaccine or whole inactivated virus vaccine. In particular embodiments, the first, second, fourth, fifth, seventh, and eighth chimeric HA polypeptides may be ones described in Section 5.1.1 below (in particular, Section 5.1.1.1 below), Section 6.1 below, Section 6.2 below, Section 6.3 below, or Section 6.4 below and the third, sixth and ninth chimeric HA polypeptide may be one described in Section 5.1.2 below or Section 6.5 below.

[0045] Also provided herein is a method of immunizing a subject against influenza virus comprising administering an immunogenic composition comprising a chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain, wherein the globular head domain is heterologous to the stem domain, wherein the liposomal adjuvant comprises liposomes and either a TLR-4 agonist, saponin or both, and wherein the composition in an animal induces influenza virus-specific T cells in the lung. See Section 5.1 below and Section 6 below for examples of chimeric HA polypeptides that may be present in the composition.

[0046] Also provided herein is an immunogenic composition comprising a chimeric influenza virus HA polypeptide and a liposomal adjuvant, for use in a method of immunizing a subject against influenza virus, wherein the method comprises administering the chimeric HA polypeptide to the subject, wherein chimeric HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain, wherein the globular head domain is heterologous to the stem domain, wherein the liposomal adjuvant comprises liposomes and either a TLR-4 agonist, saponin or both, and wherein the composition in an animal induces influenza virus-specific T cells in the lung. See Section 5.1 below and Section 6 below for examples of chimeric HA polypeptides that may be present in the composition.

[0047] In specific embodiments, an immunogenic composition described herein is administered to a subject intramuscularly. In some embodiments, an immunogenic composition described herein is administered to a subject intravenously.

[0048] In some embodiments, a subject administered an immunogenic composition described herein is a human adult. In certain embodiments, a subject administered an immunogenic composition described herein is an elderly human. In other embodiments, an immunogenic composition described herein is a human infant or human toddler.

[0049] Also provided herein is a kit comprising a container comprising an immunogenic composition described herein. In some embodiments, provided here is a kit for immunizing against an influenza virus in a subject comprising (a) a first container comprising an

immunogenic composition, wherein the immunogenic composition comprises a chimeric influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) a second container comprising a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. See Section 5.1 below and Section 6 below for examples of chimeric HA polypeptides that may be present in the composition.

[0050] In certain embodiments, the liposomal adjuvant comprises a TLR4 agonist and saponin. In certain embodiments, the TLR4 agonist is a lipopolysaccharide. In certain embodiments, the TLR4 agonist is a non-toxic derivative of lipid A. In certain embodiments, the TLR4 agonist is a monophosphoryl lipid A. In certain embodiments, the TLR4 agonist is 3-de- O-acylated monophosphoryl lipid A. In certain embodiments, the saponin is obtainable from Quillaja saponaria. In certain embodiments, the saponin comprises one or more of QS7, QS17, QS18 and QS21. In certain embodiments, the saponin comprises QS21. In certain

embodiments, the TLR4 agonist is 3-O-desacyl-monophosphoiyl lipid A and the saponin is QS21. In certain embodiments, the liposomes are 30-200 nm in size. In certain embodiments, the liposomes are 95-120 nm in size. In certain embodiments, the liposomes comprise 1,2- dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). In certain embodiments, the composition comprises a human dose volume of 0.05 ml to 1 ml of the liposomal adjuvant. In certain embodiments, the composition comprises a human dose volume of 0.4 ml to 0.6 ml of the liposomal adjuvant. In certain embodiments, the liposomal adjuvant comprises 1-100 μg of the TLR4 agonist per human dose. In certain embodiments, the liposomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose. In certain embodiments, the liposomal adjuvant comprises 20-30 μg of the TLR4 agonist per human dose. In certain embodiments, the liposomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose. In certain embodiments, the liposomal adjuvant comprises 1-100 μg of the saponin per human dose. In certain embodiments, the liposomal adjuvant comprises 20-30 μg of the TLR4 agonist and 20-30 μg of saponin per human dose. In certain embodiments, the liposomal adjuvant comprises 40-60 μg of the TLR4 agonist and 40-60 μg of saponin per human dose. In a specific embodiment, the liposomal adjuvant is one described herein (e.g., in Section 5.8.5 below or Section 6 below). In a preferred embodiment, the liposomal adjuvant is AS01 (such as described in Section 6 below).

3.1 TERMINOLOGY

[0051] As used herein, the term "120 loop" refers to an antigenic region in an influenza B virus HA. In a specific embodiment, the term "120 loop" refers to amino acid residues 116 to 137 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 116 to 137 of the HAl domain of influenza B virus B/Hong Kong/8/73 (wherein the amino acid residues 116-137 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In another specific embodiment, the term "120 loop" refers to amino acid residues 75 to 77, and 116 to 137 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 75 to 77, and 116 to 137 of the HAl domain of influenza B virus B/Hong

Kong/8/73 (wherein the amino acid residues 75 to 77 and 116-137 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In another specific embodiment, the term "120 loop" refers to amino acid residues 75, 77, and 116 to 137 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 75, 77, and 116 to 137 of the HAl domain of influenza B virus B/Hong Kong/8/73 (wherein the amino acid residues 75, 77, and 116-137 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In another specific embodiment, the term "120 loop" refers to amino acid residues 75, 77, 116, 118, 122, 129, and 137 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 75, 77, 116, 118, 122, 129, and 137 of the HAl domain of influenza B virus B/Hong Kong/8/73 (wherein the amino acid residues 75, 77, 116, 118, 122, 129, and 137 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In another specific embodiment, the term "120 loop" refers to the antigenic region defined by Wang et al, 2008, Journal of Virology 82: 3011- 3020 as 120 loop or the equivalent thereof in other influenza B viruses.

[0052] As used herein, the term "150 loop" refers to an antigenic region in an influenza B virus HA. In another specific embodiment, the term "150 loop" refers to amino acid residues 141 and 144 to 150 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 141 and 144 to 150 of the HAl domain of influenza B virus B/Hong Kong/8/73 (wherein the amino acid residues 141 and 144 to 150 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In another specific embodiment, the term "150 loop" refers to the antigenic region defined by Wang et al, 2008, Journal of Virology 82: 3011- 3020 as 150 loop or the equivalent thereof in other influenza B viruses.

[0053] As used herein, the term "160 loop" refers to an antigenic region in an influenza B virus HA. In a specific embodiment, the term "160 loop" refers to amino acid residues 162 to 167 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 162 to 167 of the HAl domain of influenza B virus B/Hong Kong/8/73 (wherein the amino acid residues 162 to 167 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In another specific embodiment, the term "160 loop" refers to the antigenic region defined by Wang et al, 2008, Journal of Virology 82: 3011-3020 as 160 loop or the equivalent thereof in other influenza B viruses.

[0054] As used herein, the term "190 helix" refers to an antigenic region in an influenza B virus HA. In a specific embodiment, the term "190 helix" refers to amino acid residues 194 to 200 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 194 to 200 of the HAl domain of influenza B virus B/Hong Kong/8/73 (wherein the amino acid residues 194 to 200 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In a specific embodiment, the term "190 helix" refers to amino acid residues 194 to 200, 205 and 238 of the HAl domain of influenza B virus B/Hong Kong/8/73 or amino acid residues in the HAl domain of an influenza B virus other than B/Hong Kong/8/73 that correspond to amino acid residues 194 to 200, 205 and 238 of the HAl domain of influenza B virus B/Hong Kong/8/73 (wherein the amino acid residues 194 to 200, 205 and 238 correspond to the numbered positions of the influenza B virus B/Hong Kong/8/73 HA not including the signal peptide, i.e., the numbering of the mature HA). In another specific embodiment, the term " 190 helix" refers to the antigenic region defined by Wang et al., 2008, Journal of Virology 82: 3011-3020 as 190 helix or the equivalent thereof in other influenza B viruses.

[0055] 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. As used herein, 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.

[0056] The term "amino acid sequence identity" has the meaning understood to a person skilled in the art. The term "amino acid identity" generally 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, including percentages of sequence identity and similarity, 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. 215:403-410, (describing NBLAST and XBLAST), Altschul et al, 1997, Nucleic Acids Res. 25:3389-3402 (describing Gapped BLAST, and PSI-Blast). Another particular example is the algorithm of Myers and Miller (1988 CABIOS 4: 11-17) which is incorporated into the ALIGN program (version 2.0) and is available as part of the GCG sequence alignment software package. Also particular is the FASTA program (Pearson W.R. and Lipman D.J., Proc. Nat. Acad. Sci. USA, 85:2444-2448, 1988), available as part of the Wisconsin Sequence Analysis Package. Additional examples include 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; and GAP, which aligns two sequences by finding a "maximum similarity" according to the algorithm of Neddleman and Wunsch (J. Mol. Biol. 48:443-354, 1970), and is preferable where the two sequences are approximately the same length and an alignment is expected over the entire length.

[0057] As used herein, the terms "chimeric influenza virus hemagglutinin polypeptide," "chimeric influenza virus HA polypeptide," "chimeric hemagglutinin polypeptide," "chimeric HA," "chimeric hemagglutinin," "chimeric HA polypeptide," and "chimeric influenza hemagglutinin polypeptide" refer to an influenza hemagglutinin that comprises an influenza virus hemagglutinin stem domain and an influenza virus hemagglutinin head domain, wherein the influenza virus hemagglutinin head domain is heterologous to the influenza virus

hemagglutinin stem domain. See, e.g., Section 5.1 below, for a discussion of chimeric influenza virus polypeptides. In certain embodiments, the influenza virus hemagglutinin head domain of a chimeric influenza virus hemagglutinin polypeptide is from a different strain or subtype of influenza virus than the influenza virus hemagglutinin stem domain. In certain embodiments, in the context of the chimeric influenza virus hemagglutinin polypeptides described herein, a heterologous influenza virus hemagglutinin head domain refers to an influenza virus

hemagglutinin head that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 5-10%, at least 10-15%, at least 10-20%, at least 15-20%, or at least 20-25% different from the homologous head (i.e., the head domain that would normally be associated with the stem domain of the chimeric influenza virus hemagglutinin polypeptide). In certain embodiments, in the context of the chimeric influenza virus hemagglutinin polypeptides described herein, a heterologous influenza virus hemagglutinin head domain refers to an influenza virus

hemagglutinin head that is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% different from the homologous head (i.e., the head domain that would normally be associated with the stem domain of the chimeric influenza virus hemagglutinin polypeptide). In certain embodiments, in the context of the chimeric influenza virus hemagglutinin polypeptides described herein, a heterologous influenza virus hemagglutinin head domain refers to an influenza virus hemagglutinin head that is 75%-95%, 75%-90%, or 75%-85% different from the homologous head (i.e., the head domain that would normally be associated with the stem domain of the chimeric influenza virus hemagglutinin polypeptide). Those of skill in the art will recognize that such a difference can be measured using approaches known in the art and described herein, e.g., comparing sequence identity or sequence homology of the head domains. In certain embodiments, in the context of the chimeric influenza virus hemagglutinin

polypeptides described herein, a heterologous influenza virus hemagglutinin head domain refers to an influenza virus hemagglutinin head that, in a hemagglutination inhibition assay, results in antisera with at least 2, at least 3, at least 4, at least 5, or at least 6 times less hemagglutination inhibition titers relative to the hemagglutination inhibition titers of the antisera raised against the homologous heads (i.e., the head domain that would normally be associated with the stem domain of the chimeric influenza virus hemagglutinin polypeptide). Those of skill in the art will recognize that such a difference can be measured using approaches known in the art and described herein (see, e.g., Section 5.9 below). Exemplary chimeric HA are described herein and in International Publication No. WO 2013/043729, International Publication No. WO

2014/099931, U.S. Publication No. 2014/0328875 and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety.

[0058] "Conservative substitution" refers to replacement of an amino acid of one class is with another amino acid of the same class. In particular embodiments, a conservative substitution does not alter the structure or function, or both, of a polypeptide. Classes of amino acids for the purposes of conservative substitution include hydrophobic (Met, Ala, Val, Leu, He), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gin, His, Lys, Arg), conformation disrupters (Gly, Pro) and aromatic (Trp, Tyr, Phe).

[0059] As described herein, the term "ectodomain" in reference to an influenza B virus HA polypeptide would be understood by one of skill in the art. In specific embodiment, the ectodomain does not include the signal peptide, the transmembrane domain, and the cytoplasmic tail domain of an influenza B virus HA. See, e.g., Table 1, Table 2 and Table 3, below, for an exemplary influenza B virus ectodomain sequence and location. In certain embodiments, the ectodomain of an influenza B virus HA polypeptide is a region of the influenza B virus HA polypeptide that aligns with the ectodomain of influenza B/Hong Kong/8/73 virus HA

ectodomain set forth in Table 1, below. In some embodiments, the ectodomain of an influenza B virus HA polypeptide is a region of the influenza B virus HA polypeptide that aligns with the ectodomain of influenza B/Malaysia/2506/04 virus HA ectodomain set forth in Table 3, below.

[0060] Table 1. Exemplary domains for influenza B/Hong Kong/8/73 virus HA.

Abbreviations: nt= nucleotide; aa=amino acid; N/A=not applicable.

Piece 1:

[0061] Table 2. Exemplary domains for mouse adapted influenza B/Malaysia/2506/20/03 HA. The full length amino acid sequence for mouse adapted influenza B/Malaysia/2506/20/03 HA may be found in SEQ ID NO: 144.

[0062] Table 3. Exemplary domains for influenza B/Malaysia/2506/20/03 HA. The full length amino acid sequence for influenza B/Malaysia/2506/20/03 HA may be found in SEQ ID NO: 152.

[0063] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of a therapy which may have a prophylactic and/or therapeutic effect(s). In certain embodiments, an "effective amount" in the context of administration of a therapy to a subject refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an influenza virus infection, disease or symptom associated therewith; (ii) reduce the duration of an influenza virus infection, disease or symptom associated therewith; (iii) prevent the progression of an influenza virus infection, disease or symptom associated therewith; (iv) cause regression of an influenza virus infection, disease or symptom associated therewith; (v) prevent the development or onset of an influenza virus infection, disease or symptom associated therewith; (vi) prevent the recurrence of an influenza virus infection, disease or symptom associated therewith; (vii) reduce or prevent the spread of an influenza virus from one cell to another cell, one tissue to another tissue, or one organ to another organ; (viii) prevent or reduce the spread of an influenza virus from one subject to another subject; (ix) reduce organ failure associated with an influenza virus infection; (x) reduce hospitalization of a subject; (xi) reduce hospitalization length; (xii) increase the survival of a subject with an influenza virus infection or disease associated therewith; (xiii) eliminate an influenza virus infection or disease associated therewith; (xiv) inhibit or reduce influenza virus replication; (xv) inhibit or reduce the entry of an influenza virus into a host cell(s); (xvi) inhibit or reduce replication of the influenza virus genome; (xvii) inhibit or reduce synthesis of influenza virus proteins; (xviii) inhibit or reduce assembly of influenza virus particles; (xix) inhibit or reduce release of influenza virus particles from a host cell(s); (xx) reduce influenza virus titer; and/or (xxi) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0064] In certain embodiments, the effective amount does not result in complete protection from an influenza virus disease, but results in a lower titer or reduced number of influenza viruses compared to an untreated subject with an influenza virus infection. In certain

embodiments, the effective amount results in a 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater reduction in titer of influenza virus relative to an untreated subject with an influenza virus infection. In some embodiments, 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 9 logs, or 8 to 9 logs. Benefits of a reduction in the titer, number or total burden of influenza virus include, but are not limited to, less severe symptoms of the infection, fewer symptoms of the infection and a reduction in the length of the disease associated with the infection.

[0065] In certain embodiments, an effective amount of a therapy (e.g., a chimeric HA polypeptide described herein or a composition thereof) results in an anti-influenza virus HA titer in a blood sample from a subject administered the effective amount 0.5 fold to 10 fold, 0.5 fold to 4 fold, 0.5 fold to 3 fold, 0.5 fold to 2 fold, 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold higher post-immunization relative to the anti -influenza virus HA titer in a blood sample from the subject prior to immunization. In certain

embodiments, an effective amount of a therapy (e.g., a chimeric HA polypeptide described herein or a composition thereof) results in an anti-influenza virus HA stalk titer in a blood sample from a subject administered the effective amount 0.5 fold to 10 fold, 0.5 fold to 4 fold, 0.5 fold to 3 fold, 0.5 fold to 2 fold, 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold higher post-immunization relative to the anti-influenza virus HA stalk titer in a blood sample from the subject prior to immunization. In certain embodiments, an effective amount of a therapy (e.g., a chimeric HA polypeptide described herein or a composition thereof) results in an anti-influenza virus neuraminidase titer in a blood sample from a subject administered the effective amount 0.5 fold to 10 fold, 0.5 fold to 4 fold, 0.5 fold to 3 fold, 0.5 fold to 2 fold, 0.5 fold, 1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold higher post-immunization relative to the anti -influenza virus neuraminidase titer in a blood sample from the subject prior to immunization.

[0066] As used herein, the term "elderly human" refers to a human 65 years or older.

[0067] The term "fragment" in the context of a nucleic acid sequence refers to a nucleotide sequence comprising a portion of consecutive nucleotides from a parent sequence. In a specific embodiment, the term refers to a nucleotide sequence of 5 to 15, 5 to 25, 10 to 30, 15 to 30, 10 to 60, 25 to 100, 50 to 100, 75 to 100, 150 to 300 or more consecutive nucleotides from a parent sequence. In another embodiment, the term refers to a nucleotide sequence of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 125, 150, 175, 200, 250, 275, 300, 325, 350, 375, 400, 425, 450 or 475 consecutive nucleotides of a parent sequence. The term "fragment" in the context of an amino acid sequence refers to an amino acid sequence comprising a portion of consecutive amino acid residues from a parent sequence. In a specific embodiment, the term refers to an amino acid sequence of 8 to 15, 10 to 20, 2 to 30, 5 to 30, 10 to 60, 25 to 100, 50 to 100, 75 to 100, 150 to 300 or more consecutive amino acid residues from a parent sequence. In another embodiment, the term refers to an amino acid sequence of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 125, 150, 175, or 200 consecutive amino acid residues of a parent sequence.

[0068] "HA" and "hemagglutinin" refer to any influenza virus hemagglutinin known to those of skill in the art or a derivative thereof. In certain embodiments, the hemagglutinin is an influenza A hemagglutinin, an influenza B hemagglutinin, or an influenza C hemagglutinin. A typical hemagglutinin comprises domains known to those of skill in the art including a signal peptide (optional herein), a stem domain, a globular head domain, a luminal domain (optional herein), a transmembrane domain (optional herein) and a cytoplasmic domain (optional herein). In certain embodiments, a hemagglutinin consists of a single polypeptide chain, such as HA0. In certain embodiments, a hemagglutinin consists of more than one polypeptide chain in quaternary association, e.g. HAl and HA2. Those of skill in the art will recognize that an immature HAO might be cleaved to release a signal peptide (generally approximately 15-20 amino acids) yielding a mature hemagglutinin HAO (i.e., HAO without a signal peptide). In the context of an influenza A virus, a mature hemagglutinin HAO might be cleaved at another site to yield HAl polypeptide (approximately 320 amino acids, including the globular head domain and a portion of the stem domain) and HA2 polypeptide (approximately 220 amino acids, including the remainder of the stem domain, a luminal domain, a transmembrane domain and a cytoplasmic domain). In the context of an influenza B virus, a mature hemagglutinin HAO might be cleaved at another site to yield HAl polypeptide (approximately 344 amino acids of influenza B/Hong Kong/8/73 virus, including the globular head domain and a portion of the stem domain) and HA2 polypeptide (approximately 223 amino acids of influenza B/Hong Kong/8/73 virus, including the remainder of the stem domain, a luminal domain, a transmembrane domain and a cytoplasmic domain). Those of skill in the art will recognize that an influenza B virus has an elongated fusion domain (composed of the central coiled-coil structure from the HA2 domain), the extended regions from HAl (amino acid residues 1-42), and HAl (amino acid residues 288-342), a globular membrane-distal domain containing the receptor-binding subdomain, HAl (amino acid residues 116-274), and a vestigial esterase subdomain, HAl (amino acid residues 43-115) and HAl (amino acid residues 275-287) (see Wang et al, 2008, Journal of Virology, 82(6):3011- 3020). Those of skill in the art will recognize that the delineation of the domains of an influenza B virus HA may be determined from, e.g., crystal structure and/or by using structure prediction software (for example, the website for the Center for Biological Sequence Analysis, Technical University of Denmark DTU, or Pymol) in conjunction with protein alignments. Thus, in one aspect, one skilled in the art will recognize that the delineation of the domains of influenza B/Hong Kong/8/73 virus HA are as set forth in Table 1, above. In another aspect, one skilled in the art will recognize the delineation of domains of the mouse adapted influenza

B/Malaysia/2506/20/03 virus HA. See, e.g., Table 2, above, for exemplary domains for the mouse adapted influenza B/Malaysia/2506/20/03 virus HA. In another aspect, one skilled in the art will recognize the delineation of domains of the influenza B/Malaysia/2506/04 virus HA. See, e.g., Table 3, above, for exemplary domains of the influenza B/Malaysia/2506/04 virus HA. In certain embodiments, a hemagglutinin comprises a signal peptide, a transmembrane domain and a cytoplasmic domain. In certain embodiments, a hemagglutinin lacks a signal peptide, i.e. the hemagglutinin is a mature hemagglutinin. In certain embodiments, a hemagglutinin lacks a transmembrane domain or cytoplasmic domain, or both. As used herein, the terms

"hemagglutinin" and "HA" encompass hemagglutinin polypeptides that are modified by post- translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage and lipid modification (e.g. S-palmitoylation).

[0069] ΉΑ2" refers to a polypeptide domain that corresponds to the HA2 domain of an influenza hemagglutinin polypeptide known to those of skill in the art. In certain embodiments, an HA2 consists of a stem domain, a luminal domain, a transmembrane domain and a cytoplasmic domain (see, e.g., Scheiffle et al, 2007, EMBO J. 16(18):5501-5508, the contents of which is incorporated by reference in its entirety). In certain embodiments, an HA2 consists of a stem domain, a luminal domain and a transmembrane domain. In certain embodiments, an HA2 consists of a stem domain and a luminal domain; in such embodiments, the HA2 might be soluble. In certain embodiments, an HA2 consists of a stem domain; in such embodiments, the HA2 might be soluble. In certain embodiments, an HA2 consists of amino acid residues 1-169 of the HA2 domain of an influenza B/Hong Kong/8/73 virus (see Wang et al, 2008, Journal of Virology 82: 3011-3020). In certain embodiments, an HA2 consists of amino acid residues 345- 567 of a mature influenza B/Hong Kong/8/73 virus HA (i.e., the numbering is determined from an influenza B/Hong Kong/8/73 virus HA that does not include the signal peptide).

[0070] The term "HAl C-terminal stem segment" refers to a polypeptide segment that corresponds to the carboxy-terminal portion of the stem domain of an influenza hemagglutinin HAl polypeptide. In the context of an influenza A virus, in certain embodiments, an HAl C- terminal stem segment consists of amino acid residues corresponding approximately to amino acids A_q through Ac term of an HAl domain. A_q is the cysteine residue in the HAl C-terminal stem segment that forms or is capable of forming a disulfide bond with a cysteine residue in an influenza A virus HAl N-terminal stem segment. Ac term or otherwise referred to herein as

HAlc-term is the C-terminal amino acid of the HAl domain as recognized by those of skill in the art. Residue A_q is identified in influenza A hemagglutinin polypeptides in Fig. 1 (i.e., A_q is Cys at amino acid position 277 of an HAl domain according to H3 numbering). Exemplary HAl C- terminal stem segments are described herein and in International Publication Nos. WO

2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety. In the context of an influenza A virus, in certain embodiments, an HAl C-terminal stem segment consists of amino acid residues corresponding approximately to amino acids 277-329 of HAl according to H3 numbering. Note that, in this numbering system, 1 refers to the N-terminal amino acid of the mature HAO protein, from which the signal peptide has been removed. Those of skill in the art will readily be able to recognize the amino acid residues that correspond to the HAl C-terminal stem segment of other influenza HA polypeptides, e.g., the amino acid residues that correspond to the HAl C-terminal stem segment of HAl from an HI hemagglutinin (see, e.g., Fig. 1). In the context of an influenza B virus, in certain embodiments, an HAl C-terminal stem segment consists of amino acid residues corresponding approximately to amino acids D_q through Ac term of an HAl domain. D_q is the alanine corresponding to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to

B/Yamagata/16/88 numbering.

[0071] The term "HAl N-terminal stem segment" refers to a polypeptide segment that corresponds to the amino-terminal portion of the stem domain of an influenza virus

hemagglutinin HAl polypeptide. In the context of an influenza A virus, in certain embodiments, an HAl N-terminal stem segment consists of amino acid residues corresponding approximately to amino acids AN-term through A_p of an HAl domain. AN-term otherwise referred to herein as HAlN-term is the N-terminal amino acid of HAl as recognized by those of skill in the art. A_p is the cysteine residue in the HAl N-terminal stem segment that forms or is capable of forming a disulfide bond with a cysteine residue in an influenza A virus HAl C-terminal stem segment. Residue A_p is identified in influenza A hemagglutinin polypeptides in Fig. 1 (i.e., A_p is Cys at amino acid position 52 of an HAl domain according to H3 numbering). Exemplary HAl N- terminal stem segments are described herein or in International Publication Nos. WO

2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety. In certain embodiments, an HAl N-terminal stem segment consists of amino acid residues corresponding approximately to amino acids 1-52 of HAl according to H3 numbering. Note that, in this numbering system, 1 refers to the N-terminal amino acid of the mature HAO protein, from which the signal peptide has been removed. Those of skill in the art will readily be able to recognize the amino acid residues that correspond to the HA1 N-terminal stem segment of other influenza HA polypeptides, e.g., the amino acid residues that correspond to the HA1 N-terminal stem segment of HA1 from an HI hemagglutinin (see, e.g., Fig. 1). In the context of an influenza B virus, in certain embodiments, an HA1 N-terminal stem segment consists of amino acid residues corresponding approximately to amino acids AN-term through D_p of an HA1 domain. D_p is the alanine corresponding to amino acid position 57 of an influenza B virus hemagglutinin HA1 domain according to B/Yamagata/16/88 numbering.

[0072] As used herein, the term "heterologous" in the context of a polypeptide, nucleic acid or virus refers to a polypeptide, nucleic acid or virus, respectively, that is not normally found in nature or not normally associated in nature with a polypeptide, nucleic acid or virus of interest. For example, a "heterologous polypeptide" may refer to a polypeptide derived from a different virus, e.g., a different influenza strain or subtype, or an unrelated virus or different species. In specific embodiments, when used in the context of a globular head domain of a chimeric influenza virus hemagglutinin described herein, the term heterologous refers to an influenza HA globular head domain that is associated with an influenza HA stem domain that it would not normally be found associated with (e.g., the head and stem domains of the HA would not be found together in nature). In specific embodiments, the heterologous HA globular head domain has a different amino acid sequence than that found normally associated with the influenza virus HA stem domain. As described above, in certain embodiments, a heterologous influenza HA globular head domain of a chimeric influenza virus hemagglutinin described herein is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 5-10%, at least 10-15%, at least 10-20%), at least 15-20%, or at least 20-25%> different from the homologous head of the hemagglutinin (i.e., the head domain that would normally be associated with the HA stem domain of the chimeric influenza virus hemagglutinin polypeptide). In certain embodiments, a heterologous influenza HA globular head domain of a chimeric influenza virus hemagglutinin described herein is at least 75%, at least 80%>, at least 85%>, at least 90%, or at least 95% different from the homologous head of the hemagglutinin (i.e., the head domain that would normally be associated with the stem domain of the chimeric influenza virus hemagglutinin polypeptide). In certain embodiments, a heterologous influenza HA globular head domain of a chimeric influenza virus hemagglutinin described herein is 75%-95%, 75%-90%, or 75%-85% different from the homologous head of the hemagglutinin (i.e., the head domain that would normally be associated with the HA stem domain of the chimeric influenza virus hemagglutinin polypeptide)). In other words, the percent identity between a heterologous influenza virus HA globular head domain of a chimeric influenza virus HA polypeptide described herein and the HA globular head domain normally associated with the HA stem domain may have less than 25%, less than 20%, less than 15%), less than 10%>, or less than 5% identity to each other.

[0073] As used herein, the term "human infant" refers to a newborn to an up to 1 year old human.

[0074] As used herein, the term "human child" refers to a human that is 1 year to up to 18 years old.

[0075] As used herein, the term "human adult" refers to a human that is 18 years or older.

[0076] As used herein, the term "in combination," in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy (e.g., more than one prophylactic agent and/or therapeutic agent). The use of the term "in combination" does not restrict the order in which therapies are administered to a subject. For example, a first therapy (e.g., a first prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.

[0077] As used herein, the term "infection" means the invasion by, multiplication and/or presence of a virus in a cell or a subject. In one embodiment, 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. In certain embodiments, 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.

[0078] As used herein, the term "influenza virus disease" refers to the pathological state resulting from the presence of an influenza (e.g., influenza A or B virus) virus in a cell or subject or the invasion of a cell or subject by an influenza virus. In specific embodiments, the term refers to a respiratory illness caused by an influenza virus.

[0079] As used herein, the terms "influenza virus hemagglutinin head domain polypeptide," "influenza virus hemagglutinin head domain," "HA globular head domain," and "HA head domain" refer to the globular head domain of an influenza hemagglutinin polypeptide known to those of skill in the art or a derivative thereof. An influenza virus hemagglutinin head domain polypeptide or influenza virus hemagglutinin head domain may comprise or consist of a known (e.g., wild-type) influenza virus hemagglutinin head domain or may comprise or consist of a derivative, e.g. an engineered derivative, of a known (e.g., wild-type) influenza virus hemagglutinin head domain. Those of skill in the art will recognize that an influenza A virus HA globular head domain typically comprises the amino acid residues intervening Cys that corresponds to amino acid position 52 of an influenza virus hemagglutinin HA1 domain according to H3 numbering and Cys that corresponds to amino acid position 277 of an influenza virus hemagglutinin HA1 domain according to H3 numbering, e.g., A_p and A_q of Figure 1, respectively. See Section 5.2 below, for information regarding influenza virus HA globular head domain polypeptides. Those of skill in the art will recognize that an influenza B virus HA globular head domain typically comprises the amino acid residues intervening Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HA1 domain according to according to B/Yamagata/16/88 numbering and Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HA1 domain according to according to B/Yamagata/16/88 numbering, e.g., D_p and D_q, respectively. Those of skill in the art will recognize that an influenza B virus HA globular head domain typically comprises the amino acid residues corresponding to amino acid residues 43-289 of the HA1 domain of influenza B/Hong Kong/8/73 virus (wherein the numbering of the amino acid residues is with respect to the mature HA sequence, which does not comprise the 15 amino acid signal peptide). For example, one skilled in the art will recognize that the amino acid sequence for the HA globular head domain for influenza B/Hong Kong/8/73 virus typically consists of the amino acid sequence:

LKGTQTRGKLCPNCLNCTDLDVALGRPKCMGTIPSAKASILHEVKPVTSGCFPIMHDR TKIRQLP LLRGYENIRLSARNVTNAETAPGGPYIVGTSGSCPNVTNGNGFFATMAWAV PK KTATNPLTVEVPYICTKGEDQITVWGFHSDDETQMVKLYGDSKPQKFTSSANGVTT HYVSQIGGFPNQAEDEGLPQSGRIVVDYMVQKPGKTGTIAYQRGVLLPQKVWCASGRS KVIKGSLPLIGE (SEQ ID NO: 122). Those of skill in the art will recognize that the location of the influenza B virus HA globular head domain for a particular strain can be determined by alignment of the influenza B virus HA polypeptide for said strain to the sequence of an influenza A virus HA (Fig. 19). In specific embodiments, the influenza B virus globular head domain consists of the amino acid residues that align to amino acid residues 58-304 of the mature influenza B/Hong Kong/8/73 virus HA (i.e., wherein said numbering includes the signal peptide). See, e.g., Table 1, above.

[0080] As used herein, the phrases "IFN deficient system" or "IFN-deficient substrate" refer to systems, e.g., cells, cell lines and animals, such as pigs, mice, chickens, turkeys, rabbits, rats, etc., which do not produce interferon (IFN) or produce low levels of IFN (i.e., a reduction in IFN expression of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more when compared to IFN-competent systems under the same conditions), do not respond or respond less efficiently to IFN, and/or are deficient in the activity of one or more antiviral genes induced by IFN.

[0081] As used herein, the numeric term "log" refers to logio.

[0082] As used herein, the phrase "multiplicity of infection" or "MOI" is the average number of infectious virus particles per infected cell. The MOI is determined by dividing the number of infectious virus particles added (ml added x PFU/ml) by the number of cells added (ml added x cells/ml). The MOI may be determined by dividing the number of virus added (ml added x Pfu/ml) by the number of cells added (Pfu added /cells).

[0083] As used herein, the term "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.

[0084] "Polypeptide" refers to a polymer of amino acids linked by amide bonds as is known to those of skill in the art. As used herein, the term can refer to a single polypeptide chain linked by covalent amide bonds. The term can also refer to multiple polypeptide chains associated by non-covalent interactions such as ionic contacts, hydrogen bonds, Van der Waals contacts and hydrophobic contacts. Those of skill in the art will recognize that the term includes polypeptides that have been modified, for example by post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage and lipid modification (e.g. S-palmitoylation). [0085] As used herein, the term "premature human infant" refers to a human infant born at less than 37 weeks of gestational age.

[0086] As used herein, the terms "prevent," "preventing" and "prevention" in the context of the administration of a therapy(ies) to a subject to prevent an influenza virus disease refer to one or more of the prophylactic/beneficial effects that may result from the administration of a therapy or a combination of therapies. In a specific embodiment, the terms "prevent," "preventing" and "prevention" in the context of the administration of a therapy(ies) to a subject to prevent an influenza virus disease refer to one or more of the following effects resulting from the administration of a therapy or a combination of therapies: (i) the inhibition of the development or onset of an influenza virus disease or a symptom thereof; (ii) the inhibition of the recurrence of an influenza virus disease or a symptom associated therewith; and (iii) the reduction or inhibition in influenza virus infection and/or replication.

[0087] As used herein, 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. Thus, a polypeptide that is isolated includes preparations of a polypeptide having less than about 30%, 20%, 10%), 5%, 2%, or 1%> (by dry weight) of cellular materials and/or contaminating materials. As used herein, the terms "purified" and "isolated" when used in the context of a polypeptide (including an antibody) that is chemically synthesized refers to a polypeptide which is substantially free of chemical precursors or other chemicals which are involved in the syntheses of the polypeptide. In a specific embodiment, a chimeric HA polypeptide is chemically synthesized. In another specific embodiment, a chimeric influenza hemagglutinin polypeptide is isolated.

[0088] As used herein, the terms "replication," "viral replication" and "virus replication" in the context of a virus refer 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. In some embodiments, the terms "replication," "viral replication" and "virus replication" refer to the replication of the viral genome. In other embodiments, the terms "replication," "viral replication" and "virus replication" refer to the synthesis of viral proteins.

[0089] As used herein, the terms "stem domain polypeptide", "influenza virus hemagglutinin stem domain polypeptide", "stalk domain" and "stalk" refer to any influenza virus hemagglutinin stem domain known to those of skill in the art or a derivative thereof, e.g. an engineered derivative, that comprises one or more polypeptide chains that make up a stem domain of hemagglutinin. A stem domain polypeptide might be a single polypeptide chain, two

polypeptide chains or more polypeptide chains. Typically, a stem domain polypeptide is a single polypeptide chain (i.e. corresponding to the stem domain of a hemagglutinin HAO polypeptide) or two polypeptide chains (i.e. corresponding to the stem domain of a hemagglutinin HA1 polypeptide in association with a hemagglutinin HA2 polypeptide). In a particular embodiment, a stem domain comprises an N-terminal HA1 stem segment, a C-terminal HA1 stem segment, and a portion of an HA2 domain (e.g., a portion of an HA2 domain that does not include the transmembrane and cytoplasmic domains; sometimes referred to as an ΉΑ2 stem domain"). One skilled in the art will understand that the exact location of the C-terminus of the HA stem domain is determined according to the hydrophobicity of the HA2 domain of the particular influenza virus HA strain and can be identified using programs such as, e.g., the TMHMM server (www. cb s . dtu . dk/servi ce s/TMHMM/; see, e.g., Cuthbertson et al., 2005, Protein Eng Des Sel, 18(6):295-308) hydrophobicity prediction, or uniprot. For example, the underlined amino acid residues in the following sequence correspond to a stem domain comprising an N-terminal HA1 stem segment, a C-terminal HA1 stem segment, and a portion of an HA2 domain that does not include the transmembrane domain nor the cytoplasmic domain, wherein the C-terminus of the stem domain is determined using the TMHMM server (www . cb s . dtu . d k servi ces/TMHMM/; see, e.g., Cuthbertson et al., 2005, Protein Eng Des Sel, 18(6):295-308) hydrophobicity prediction: MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKL CRLKGIAPLQLGKCNIAGWLLG PECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELR EQLSSVS SFERFEIFPKES SWP HNTNGVTAACSHEGKS SF YR LLWLTEKEGS YPKLKN SYVmKGKEVLVLWGIHHPPNSKEQQ LYQ ENAYVSVVTSNY RRFTPEIAERPKVR DQAGRMNYYWTLLKPGDTIIFEANG LIAPMYAFALSRGFGSGIITSNASMHECNTKCQ TPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRN PSIQSRGLFGAIAGFIEGGW TGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNK LE RMENLN VDDGFLDIWTYNAELLVLLENERTLDFHDSNV NLYE V SQL NN AKEIGNGCFEFYHKCD ECMESVRNGTYDYPKYSEESKL REKVDGVKLESMGIYQIL AIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI (SEQ ID NO: l). Alternatively, as another example, the italicized amino acid residues in the following sequence correspond to a stem domain comprising an N-terminal HA1 stem segment, a C-terminal HA1 stem segment, and a portion of an HA2 domain that does not include the transmembrane domain nor the cytoplasmic domain, wherein the C-terminus of the stem domain is determined using uniprot: MKA LLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKL CRLKGIAPLQLGKCNIAGWLLG PECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELR EQLSSVS SFERFEIFPKES SWPNHNTNGVTAACSHEGKS SF YRNLLWLTEKEGS YPKLKN SYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVR DQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQ TPLGArNS SLP YQNIHP VTIGECPK YVRS AKLRM VTGLRNNP SIQ SRGLFGAIA GFIEGG W TGMIDG WYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTA VGKEFNKLEKR MENLNKKVDDGFLDIWTYNAELL VLLENER TLDFHDSNVKNL YEKVKSQLKNNAKEIGNGCF EFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTV ASSLVLL VSLGAISFWMCSNGSLQCRICI (SEQ ID NO: l). In certain embodiments, 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. Engineered stem domain polypeptides can comprise one or more linkers as described below. See Section 5.3 below, for information regarding influenza virus HA stem domain polypeptides. See, e.g., Table 1 and Table 2 above, for an example of the amino acid sequence and location of a stem domain of an influenza B virus.

[0090] Those of skill in the art will recognize that an influenza A 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 HAN-term through A_p of an influenza virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues A_q through HAc-term of an influenza virus hemagglutinin HA1 domain, wherein HAN-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 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 domain of an H3 hemagglutinin according to H3 numbering. Those of skill in the art will recognize that an influenza B 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 HAN-term through D_p of an influenza B virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues D_q through HAc-term of an influenza B virus

hemagglutinin HA1 domain, wherein HAN-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 HA1 domain, wherein Dp is the alanine corresponding to amino acid position 57 of an influenza B virus hemagglutinin HA1 domain according to B/Yamagata/16/88 numbering, and wherein D_q is the alanine corresponding to amino acid position 306 of an influenza B virus hemagglutinin HA1 domain according to B/Yamagata/16/88 numbering.

[0091] As used herein, terms "subject" or "patient" are used interchangeably to refer to an animal (e.g., birds, reptiles, and mammals). In a specific embodiment, a subject is a bird. In another embodiment, a subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g. , a monkey, chimpanzee, and a human). In certain embodiments, a subject is a non-human animal. In some embodiments, a subject is a farm animal or pet. In another embodiment, a subject is a human. In another embodiment, a subject is a human infant. In another embodiment, a subject is a human child. In another embodiment, a subject is a human adult. In another embodiment, a subject is an elderly human. In another embodiment, a subject is a premature human infant.

[0092] As used herein, the term "seasonal influenza virus strain" refers to a strain of influenza virus to which a subject population is exposed to on a seasonal basis. In specific embodiments, the term seasonal influenza virus strain refers to a strain of influenza A virus. In specific embodiments, the term seasonal influenza virus strain refers to a strain of influenza virus that belongs to the HI or the H3 subtype, i.e., the two subtypes of influenza A virus that presently persist in the human subject population. In other embodiments, the term seasonal influenza virus strain refers to a strain of influenza B virus. In specific embodiments, the term seasonal influenza virus strain refers to a strain of influenza B virus. In specific embodiments, the term seasonal influenza virus strain refers to a strain of influenza virus that belongs to the Yamagata or the Victoria lineages, i.e., the two influenza B virus lineages that presently persist in the human subject population.

[0093] The terms "tertiary structure" and "quaternary structure" have the meanings understood by those of skill in the art. Tertiary structure refers to the three-dimensional structure of a single polypeptide chain. Quaternary structure refers to the three dimensional structure of a polypeptide having multiple polypeptide chains.

[0094] As used herein, 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. In certain embodiments, 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. In some embodiments, the term "therapy" refers to (i) a nucleic acid encoding a chimeric influenza virus hemagglutinin polypeptide, (ii) a chimeric influenza virus hemagglutinin polypeptide, or (iii) a vector or composition comprising a nucleic acid encoding a chimeric influenza virus hemagglutinin polypeptide and/or comprising a chimeric HA polypeptide. In some embodiments, the term "therapy" refers to an antibody that specifically binds to a chimeric influenza virus

hemagglutinin polypeptide.

[0095] As used herein, 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 so that a beneficial or therapeutic effect of a therapy or a combination of therapies may be obtained. In specific embodiments, such terms refer to one, two, three, four, five or more of the following effects resulting from the administration of a therapy or a combination of therapies: (i) the reduction or amelioration of the severity of an influenza virus infection or a disease or a symptom associated therewith; (ii) the reduction in the duration of an influenza virus infection or a disease or a symptom associated therewith; (iii) the regression of an influenza virus infection or a disease or a symptom associated therewith; (iv) the reduction of the titer of an influenza virus; (v) the reduction in organ failure associated with an influenza virus infection or a disease associated therewith; (vi) the reduction in hospitalization of a subject; (vii) the reduction in hospitalization length; (viii) the increase in the survival of a subject; (ix) the elimination of an influenza virus infection or a disease or symptom associated therewith; (x) the inhibition of the progression of an influenza virus infection or a disease or a symptom associated therewith; (xi) the prevention of the spread of an influenza virus from a cell, tissue, organ or subject to another cell, tissue, organ or subject; (xii) the inhibition or reduction in the entry of an influenza virus into a host cell(s); (xiii) the inhibition or reduction in the replication of an influenza virus genome; (xiv) the inhibition or reduction in the synthesis of influenza virus proteins; (xv) the inhibition or reduction in the release of influenza virus particles from a host cell(s); and/or (xvi) the enhancement or improvement the therapeutic effect of another therapy.

[0096] As used herein, in some embodiments, the phrase "wild-type" in the context of a viral polypeptide refers to a viral polypeptide that is found in nature and is associated with a naturally occurring virus.

[0097] As used herein, in some embodiments, the phrase "wild-type" in the context of a virus refers to the types of a virus that are prevalent, circulating naturally and producing typical outbreaks of disease. In other embodiments, the term "wild-type" in the context of a virus refers to a parental virus.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] FIGS. 1A-1D. Sequence alignment by CLUSTALW of representative sequences of 17 subtypes of influenza virus A hemagglutinin (SEQ ID NOS: 1-17, H1-H17, respectively). The residue designated Ap is the cysteine residue in the HA1 N-terminal stem segment that forms or is capable of forming a disulfide bond with the residue designated Aq, a cysteine residue in an HA1 C-terminal stem segment. Due to size limitations, the sequence alignment is split between Fig. 1A, Fig. IB, Fig. 1C and Fig. ID.

[0099] FIG. 2 depicts the study layout. D: day; LD50: lethal dose, 50%; HlNlpdm09 is A/Netherlands/602/2009. [00100] FIGS. 3A-3C. T cell responses post vaccination measured in the spleen. Spleens were harvested 10 days post final vaccination and stimulated either with a CD8-restricted P epitope (AS E METM (SEQ ID NO: 100)) from PR8 (FIG. 3 A), whole HlNlpdm09 virus (FIG. 3B) or an irrelevant peptide (FIG. 3C). Results from individual mice are shown as points. White bars indicate the geometric mean for each group. Key: Filled circles (QIV - IIV - IIV (AS01)); filled squares (QIV - IIV - IIV (AS03)); empty circles (QIV - IIV - IIV (non- adjuvanted)); triangles pointing up (QIV - LAIV - mock); diamonds (QIV - mock - LAIV); circles with lines inside (QIV - LAIV - IIV (AS03); triangles pointing down (QIV - IIV - LAIV (AS03); stars (QIV - LAIV - IIV (non-adjuvanted)); squares (mock - mock - mock).

[00101] FIGS. 4A-4C. T cell responses post vaccination measured in the lung. Lungs were harvested 10 days post final vaccination and stimulated either with a CD 8 -restricted NP epitope from PR8 (ASNENMETM (SEQ ID NO: 100)) (FIG. 4A), whole HlNlpdm09 virus (FIG. 4B), or an irrelevant peptide (FIG. 4C). Results from individual mice are shown as points. White bars indicate the geometric mean for each group. Key: Filled circles (QIV - IIV - IIV (AS01)); filled squares (QIV - IIV - IIV (AS03)); empty circles (QIV - IIV - IIV (non-adjuvanted)); triangles pointing up (QIV - LAIV - mock); diamonds (QIV - mock - LAIV); circles with lines inside (QIV - LAIV - IIV (AS03); triangles pointing down (QIV - IIV - LAIV (AS03); stars (QIV - LAIV - IIV (non-adjuvanted)); squares (mock - mock - mock).

[00102] FIGS. 5A-5E. T cell responses post challenge measured in the spleen. Spleens were harvested 6 days post challenge and stimulated either with a CD8-restricted NP epitope from PR8 (ASNENMETM (SEQ ID NO: 100)) (FIG. 5A), NP overlap pool peptide (FIG. 5B), whole HlNlpdm09 virus (FIG. 5C), HA overlap pool peptide (FIG. 5D), or an irrelevant peptide (FIG. 5E). Results from individual mice are shown as points. White bars indicate the geometric mean for each group. Key: Filled circles (QIV - IIV - IIV (AS01)); filled squares (QIV - IIV - IIV (AS03)); empty circles (QIV - IIV - IIV (non-adjuvanted)); triangles pointing up (QIV - LAIV - mock); diamonds (QIV - mock - LAIV); circles with lines inside (QIV - LAIV - IIV (AS03); triangles pointing down (QIV - IIV - LAIV (AS03); stars (QIV - LAIV - IIV (non- adjuvanted)); squares (mock - mock - mock).

[00103] FIGS. 6A-6D. T cell responses post challenge measured in the lung. Right lungs were harvested 6 days post challenge and stimulated either with a CD8-restricted NP epitope from PR8 (ASNENMETM (SEQ ID NO: 100)) (FIG. 6A), whole HlNlpdm09 virus (FIG. 6B), HA overlap pool peptide (FIG. 6C), or an irrelevant peptide (FIG. 6D). Results from individual mice are shown as points. White bars indicate the geometric mean for each group. Key: Filled circles (QIV - IIV - IIV (AS01)); filled squares (QIV - IIV - IIV (AS03)); empty circles (QIV - IIV - IIV (non-adjuvanted)); triangles pointing up (QIV - LAIV - mock); diamonds (QIV - mock - LAIV); circles with lines inside (QIV - LAIV - IIV (AS03); triangles pointing down (QIV - IIV - LAIV (AS03); stars (QIV - LAIV - IIV (non-adjuvanted)); squares (mock - mock - mock).

[00104] FIGS. 7A-FIG. 7B. Viral lung titers post challenge and HI stalk ELISA antibody titers. FIG. 7A: Left lungs were collected 6 days post challenge and lung viral titers were quantified through plaque assays. Results from individual mice are shown as points. White bars indicate the geometric mean for each group. FIG. 7B: Anti-Hl stalk antibody levels in serum collected post 1st boost, 2nd boost and challenge were measured through ELISA. Results from pooled serum from each group are shown as points. Key: Filled circles (QIV - IIV - IIV (AS01)); filled squares (QIV - IIV - IIV (AS03)); empty circles (QIV - IIV - IIV (non- adjuvanted)); triangles pointing up (QIV - LAIV - mock); diamonds (QIV - mock - LAIV); circles with lines inside (QIV - LAIV - IIV (AS03); triangles pointing down (QIV - IIV - LAIV (AS03); stars (QIV - LAIV - IIV (non-adjuvanted)); squares (mock - mock - mock).

[00105] FIG. 8. Anti-Hl stalk domain IgG ELISA titers in sera from mice following a two- dose regimen of cH8/l and cH5/l without adjuvant or formulated with AS03A or ASOle-derived in QlV-primed mice. Forty BALB/c mice were used for the cHA and QIV groups, and twenty mice were used for the PBS control group. IgG antibody titers directed towards HI -stalk domain were measured by ELISA assay on pooled sera (technical triplicates were performed) using a cH6/l recombinant antigen. For each time point, ELISA endpoint values are shown. Titers are expressed as arithmetic means of the technical triplicates.

[00106] FIG. 9. Anti-Hl stalk domain IgG ELISA titers at day 84 in sera from individual mice following a two-dose regimen of cH8/l and cH5/l without adjuvant or formulated with AS03A or ASOlB-derived in QlV-primed mice. Forty BALB/c mice were used for the cHA and QIV groups, and twenty mice were used for the PBS control group. IgG antibodies titers directed towards the HI -stalk were measured by an ELISA assay on individual sera from day 84 using a cH6/l recombinant antigen. ELISA endpoint values are shown. Bars represent geometric means and the error bars indicate 95% CI. The asterisks refer to the significance of the p-value. Symbol meaning *: p< 0.0001.

[00107] FIGS. lOA-lOC. Cross-reactive anti-HA IgG antibody titers in sera following a two- dose regimen with, cH8/l and cH5/l without adjuvant or formulated with AS03A or ASO IB- derived in QIV primed mice. Forty BALB/c mice were used for the cHA and QIV groups, and twenty mice were used for the PBS control group. IgG antibodies directed towards full-length HA antigen H2 (FIG. 10), H9 (FIG. 10B) or HI 8 (FIG. I OC) were measured by ELISA assay on pooled sera. For each time point, ELISA endpoint values are shown. Titers are expressed as arithmetic means of the technical triplicates.

[00108] FIG. 11. Anti-Nl cross-reactive IgG ELISA titers in sera from mice following a two- dose regimen with, cH8/l and cH5/l without adjuvant or formulated with AS03A or ASO IB- derived in QIV primed mice. Forty BALB/c mice were used for the cHA and QIV groups, and twenty mice were used for the PBS control group. IgG antibodies directed against Nl antigen were measured by ELISA assay on pooled sera. For each time point, ELISA endpoint values are shown. Titers are expressed as arithmetic means of the technical triplicates.

[00109] FIGS. 12A-12B. ADCC activity against A/HlNlpdm09 at day 84 following a two- dose regimen with, cH8/l and cH5/l without adjuvant or formulated with AS03A or ASO IB- derived in QIV primed mice. Forty BALB/c mice were used for the cHA and QIV groups, and twenty mice were used for the PBS control group. Fc-mediated effector activity against

HlNlpdm09 was measured in pooled serum (technical duplicates) using an ADCC-reporter in vitro assay. ADCC activities are expressed as luminescence per serum dilution (FIG. 12A) or area under the curve (FIG. 12B).

[00110] FIGS. 13A-13B. ADCC activity against cH6/lN5 at Day 84 following a two-dose regimen with, cH8/l and cH5/l without adjuvant or formulated with AS03A or ASOlB-derived in QIV primed mice. Forty BALB/c mice were used for the cHA and QIV groups, and twenty mice were used for the PB S control group. Fc-mediated effector activity against cH6/lN5 was measured in pooled serum (technical duplicates) using an ADCC-reporter in vitro assay. ADCC activities are expressed as luminescence per serum dilution (FIG. 13 A) or area under the curve (FIG. 13B).

[00111] FIGS. 14A-14B. Mouse weight loss and survival curves after challenge with

CH6/1N5. Forty BALB/c mice were used for each formulation groups, and twenty mice in the PBS control group. Group-pooled sera from vaccinated mice at day 84 were transferred into naive BALB/c mice (10 BALB/c mice six-to-eight-week-old female per transfer group, 150μ1 of serum per mouse). Two to five hours after the serum transfer, the mice were challenged with 10xLD50 (200 PFU) of cH6/lN5 virus. Weight lost (FIG. 14A) and survival (FIG. 14B) were monitored daily. For weight loss, each dot represents the mean (with standard error of mean). The horizontal dash line represents the weight loss ethical endpoint limit (25% of initial body weight loss).

[00112] FIGS. 15A-15B. Mouse weight loss and survival curves after challenge with

A/HlNlpdm09. Pooled sera from vaccinated mice or from PBS control mice at day 84 were transferred into naive BALB/c mice (10 BALB/c mice six-to-eight-week-old female per transfer group, 150μ1 of serum per mouse). Two to five hours after the serum transfer, the mice were challenged with 10xLD50 (200 PFU) of A/HlNlpdm09 virus. Weight lost (FIG. 15A) and survival (FIG. 15B) were monitored daily. For weight loss, each dot represents the mean (with standard error of mean). The horizontal dash line represents the weight loss ethical endpoint limit (25% of initial body weight loss).

[00113] FIG. 16. Anti-Hl stalk domain IgG ELISA titers in sera from QlV-primed rabbits. Rabbits were immunized on D 0, D 21, and D 35 with QIV, CH8/1N1 and CH5/1N1,

respectively. Serum samples were collected on D 0, D 21, D 38 (3 D post-third immunization), and D 63 (28 D post-third immunization). Four rabbits (two of each gender) were included in each group: non-adjuvanted (squares), adjuvanted with ASOlB-derived (triangles), or adjuvanted with AS03 A (circles). IgG antibodies directed towards the HI stalk domain were measured by ELISA assay using cH6/l recombinant antigen. For each timepoint, ELISA Unit values are represented. Titers are expressed as geometric means +/- 95% confidence interval (CI).

[00114] FIG. 17. Anti-Hl stalk domain IgG ELISA titers in sera from unprimed rabbits. Rabbits were immunized at D 21 and D 35 with CH8/1N1 and CH5/1N1, respectively. Serum samples were collected on D 0, D 38 (3 D post-second immunization), and D 63 (28 D post- second immunization). Four rabbits (two of each gender) were included in each group: non- adjuvanted (squares), adjuvanted with ASOlB-derived (triangles), or adjuvanted with AS03A (circles). IgG antibodies directed towards the HI stalk domain were measured by ELISA assay using cH6/l recombinant antigen. For each timepoint, ELISA Unit values are represented.

Titers are expressed as geometric means +/- 95% confidence interval (CI). [00115] FIG. 18. Study design.

[00116] FIG. 19: Alignment of influenza B/Hong Kong/8/73 virus HA (SEQ ID NO: 169), influenza A/Puerto Rico/8/34 virus HA (SEQ ID NO: 170), and influenza B/Yamagata/16/88 virus HA (SEQ ID NO: 171). The locations of the signal peptide, stalk domain, head domain, start of the HA2 domain, fusion peptide, transmembrane domain, and cytoplasmic tail domain for the influenza B viruses are delineated based on the locations of the respective domains in the influenza A virus.

[00117] FIGS. 20A-20B. FIG. 20 A: Nucleic acid sequence encoding influenza

A/mallard/Sweden/24/2002 virus HA (SEQ ID NO: 172). Underlined sequences are the 5' and 3' noncoding regions. FIG. 20B: Amino acid sequence (SEQ ID NO: 173) of the nucleic acid of Fig. 20 A.

[00118] FIGS. 21A-21B. FIG. 21 A: Nucleic acid sequence encoding influenza

A/Vietnam/1203/04 (HALo) virus HA (SEQ ID NO: 174). FIG. 21B: Amino acid sequence (SEQ ID NO: 175) of the nucleic acid of Fig. 21 A.

[00119] FIGS. 22A-22B. FIG 22 A: Nucleic acid sequence encoding influenza A/northern shoveler/Netherlands/18/99 virus HA (SEQ ID NO: 176). Underlined sequences are the 5' and 3' non-coding regions. FIG. 22B: Amino acid sequence (SEQ ID NO: 177) of the nucleic acid of Fig. 22 A.

[00120] FIGS. 23A-23B. FIG. 23A: Nucleic acid sequence encoding A mallard interior Alaska_7MP0167_2007 virus HA (SEQ ID NO: 178). Underlined sequences are the 5' and 3' non-coding regions. FIG. 23B: Amino acid sequence (SEQ ID NO: 179) of the nucleic acid of Fig. 23 A.

[00121] FIGS. 24A-24B. FIG. 24A: Nucleic acid sequence encoding influenza A/Puerto Rico/8/34 virus HA (SEQ ID NO: 180). Underlined sequences are the 5' and 3' non-coding regions. FIG. 24B: Amino acid sequence (SEQ ID NO: 181) of the nucleic acid of Fig. 24A.

[00122] FIGS. 25A-25B. FIG. 25A: Nucleic acid sequence encoding influenza

B/Yamagata/16/88 virus HA (SEQ ID NO: 182). Underlined sequences are the 5' and 3' non- coding regions. FIG. 25B: Amino acid sequence (SEQ ID NO: 183) of the nucleic acid of FIG. 25A. [00123] FIG. 26 depicts the four major antigenic sites of influenza B virus HA in the influenza virus B/Yamanashi/166/1988 (PDB: 4M40): 120 loop, 150 loop, 160 loop, and 190 helix.

[00124] FIG. 27: Amino acid residues in four antigenic sites (120 loop, 150 loop, 160 loop, and 190 helix) of the influenza B/Yamagata/16/88 virus HA were replaced by corresponding amino acid sequences from influenza A virus HAs of the H5, H8, HI 1 or HI 3 subtypes. The resulting constructs are referred to herein as mH5/B, mH8/B, mHl 1/B, and mH13/B,

respectively, chimeric HAs. Viruses encoding the chimeric HAs were rescued in an influenza B/Malaysia/2506/04 MA virus backbone.

[00125] FIG. 28: Nucleic acid sequence encoding mH5/B chimeric HA based on an influenza B/Yamagata/16/88 virus HA sequence, comprising nucleic acid sequences from the influenza A/Vietnam/1203/04(HALo) virus (H5) globular head domain (SEQ ID NO: 184). Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain. Uppercase bold sequences correspond to additional mutations introduced into the influenza B/Yamagata/16/88 virus HA ectodomain.

[00126] FIG. 29: Amino acid sequence (SEQ ID NO: 185) encoded by the nucleic acid of FIG. 28. Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain. Uppercase bold sequence corresponds to an additional mutation introduced into the influenza B/Yamagata/16/88 virus HA ectodomain.

[00127] FIG. 30: Nucleic acid sequence encoding mH8/B chimeric HA based on an influenza B/Yamagata/16/88 virus HA sequence, comprising nucleic acid sequences from the influenza A/Mallard/Sweden/24/2002 virus (H8) globular head domain (SEQ ID NO: 186). Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain. Uppercase bold sequences correspond to additional mutations introduced into the influenza B/Yamagata/16/88 virus HA ectodomain.

[00128] FIG. 31: Amino acid sequence (SEQ ID NO: 187) encoded by the nucleic acid of FIG. 30. Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain. Uppercase bold sequence corresponds to an additional mutation introduced into the influenza B/Yamagata/16/88 virus HA ectodomain.

[00129] FIG. 32: Nucleic acid sequence encoding mHl 1/B chimeric HA based on an influenza B/Yamagata/16/88 virus HA sequence, comprising nucleic acid sequences from the influenza A/northern shovel er/Netherlands/18/99 virus (HI 1) globular head domain (SEQ ID NO: 188). Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain. Uppercase bold sequence corresponds to an additional mutation introduced into the influenza B/Yamagata/16/88 virus HA ectodomain.

[00130] FIG. 33: Amino acid sequence (SEQ ID NO: 107) encoded by the nucleic acid of FIG. 32. Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain. Uppercase bold sequence corresponds to an additional mutation introduced into the influenza B/Yamagata/16/88 virus HA ectodomain.

[00131] FIG. 34: Nucleic acid sequence encoding mH13/B chimeric HA based on an influenza B/Yamagata/16/88 virus HA sequence, comprising nucleic acid sequences from the influenza A/black headed gull/Sweden/ 1/99 (H13) globular head domain (SEQ ID NO: 140). Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain.

[00132] FIG. 35: Amino acid sequence (SEQ ID NO: 141) encoded by the nucleic acid of FIG. 34. Bold, underlined, uppercase sequences correspond to mutations introduced into the 120 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Bold lowercase sequences correspond to mutations introduced into the 150 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Italicized, underlined lowercase sequences correspond to mutations introduced into the 160 loop of the influenza B/Yamagata/16/88 virus HA ectodomain. Underlined, bold, italicized, uppercase sequences correspond to mutations introduced into the 190 helix of the influenza B/Yamagata/16/88 virus HA ectodomain.

[00133] FIGS. 36A-36D: depicts growth curves for influenza viruses expressing mH5/B chimeric HA (FIG. 36A), mH8/B chimeric HA (FIG. 36B), mHl 1/B chimeric HA (FIG. 36C), or mH13/B chimeric HA (FIG. 36D) as compared to wild type influenza B/Malaysia/2506/04 MA virus. 10-day embryonated eggs were infected with 500 PFU/egg of the influenza virus expressing mH5/B chimeric HA (mH5/B Mai), mH8/B chimeric HA (mH8/B Mai), mHl 1/B chimeric HA (mHl 1/B Mai), or mH13/B chimeric HA (mH13/B Mai), or wild type influenza B/Malaysia/2506/04 MA vims (B/Mal04 MA) in triplicates and incubated at 33 degrees Celsius. Allantoic fluids were harvested at the indicated times and plaque assays were performed on Madin Darby Canine Kidney (MDCK) cells to determine virus titers. PFU refers to plaque forming unit. B/Mal04 MA refers to wild type influenza B/Malaysia/2506/04 MA virus. mH5/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH5/B chimeric HA described in FIGS. 28 and 29. mH8/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH8/B chimeric HA described in FIGS.30 and 31. mHl 1/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mHl 1/B chimeric HA described in FIGS. 32 and 33. mH13/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH13/B chimeric HA described in FIGS. 34 and 35.

[00134] FIGS. 37A-37D demonstrate that cross-protective subdominant conserved antigenic sites within influenza B virus HA were preserved in the chimeric HA. MDCK cells were infected with influenza viruses expressing mH5/B chimeric HA (FIG. 37 A), mH8/B chimeric HA (FIG. 37B), mHl 1/B chimeric HA (FIG. 37C), or mH13/B chimeric HA (FIG. 37D) at an MOI of 5 without TPCK-trypsin and compared to uninfected cells or cells infected with B/Mal04 MA at an MOI of 5 without TPCK-trypsin. Cells were incubated at 33 degrees Celsius and 5% CO2. 17 hours post infection, cells were fixed with methanol free 5% paraformaldehyde for immunofluorescence surface staining using the indicated anti-influenza B virus HA cross- protective human/mouse monoclonal antibodies and anti -influenza B virus HA polyclonal mouse serum. Secondary Alexa Fluor anti-human or anti-mouse antibody were used. Images were taken using Zeiss LSM 880 confocal microscope. PFU refers to plaque forming unit. B/Mal04 MA refers to wild type influenza B/Malaysia/2506/04 MA virus. mH5/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH5/B chimeric HA described in FIGS. 28 and 29. mH8/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH8/B chimeric HA described in FIGS. 30 and 31. mHl 1/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mHl 1/B chimeric HA described in FIGS. 32 and 33. mH13/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH13/B chimeric HA described in FIGS. 34 and 35. TPCK refers to L-l-Tosylamide-2-phenylethyl chloromethyl ketone.

[00135] FIGS. 38A-38D demonstrate that immunodominant epitopes on influenza

B/Yamagata/88 HA head were ablated in the chimeric HAs. Mouse and ferret sera were raised against wild type influenza B virus strain B/Yamagata/ 16/88 to acquire hemagglutination inhibition (HI) reactivity. HI assays for the mouse and ferret sera were performed using turkey red blood cells (RBCs) with influenza viruses expressing mH5/B chimeric HA (FIG. 38 A), mH8/B chimeric HA (FIG. 38B), mHl 1/B chimeric HA (FIG. 38C), or mH13/B chimeric HA (FIG. 57D) or wild type influenza B virus strain B/Yamagata/16/88 (FIGS. 38A-38D). B Yamagata 88 wild type refers to wild type influenza B/Yamagata/16/88 virus. mH5/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH5/B chimeric HA described in FIGS. 28 and 29. mH8/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH8/B chimeric HA described in FIGS. 30 and 31. mHl 1/B Mai refers to influenza

B/Malaysia/2506/04 MA virus encoding the mHl 1/B chimeric HA described in FIGS. 32 and 33. mH13/B Mai refers to influenza B/Malaysia/2506/04 MA virus encoding the mH13/B chimeric HA described in FIGS. 34 and 35.

[00136] FIGS. 39A and 39B depict weight loss (FIG. 39A) and survival (FIG. 39B) of chimeric HA-vaccinated mice after challenge with B/Malaysia/2506/04 (Victoria-like).

Vaccination with the chimeric HA regiment resulted in complete protection from mortality with minimal weight loss. Circles: Group 1 (chimeric HA); squares: Group 2 (prime only); triangles pointing up: Group 3 (TIV); triangles pointing down: Group 4 (irrelevant protein); diamonds: naive.

[00137] FIGS. 40A-40B. FIG. 40A: Nucleic acid sequence encoding influenza A/black headed gull/Sweden/ 1/99 virus HA (SEQ ID NO: 142). FIG. 40B: Amino acid sequence (SEQ ID NO: 143) of the nucleic acid of Fig. 40A.

[00138] FIGS. 41A-41C: demonstrates that AS01 adjuvanted cHA vaccination elicits higher IgG2a titers compared to AS03 (indicative of Thl response). Day 84 serum cH6/l IgG subtype ELISA titers for HI stalk IgGi antibodies (FIG. 41 A) and HI stalk IgG_2a antibodies (FIG. 41B) are provided. FIG. 41C provides the ratio of HI stalk IgG_2a/IgGi antibody titers from FIG. 41A and FIG. 41B.

[00139] FIG. 42A and FIG. 42B: depicts that H9 cross-reactive antibody responses are affected by sequence of cHA dosing. FIG. 42 A: ELISA of H9 cross-reactive antibodies at the indicated dates for the indicated vaccination regimens (pool sera, technical triplicates). FIG. 42B: ELISA of H9 antibodies post-vaccination (D84) (individual mouse sera).

[00140] FIG. 43A-43F. FIG. 43 A: HI stalk IgGl antibody titers (pooled sera). FIG. 43B: HI stalk IgGl antibodies (D84) (individual mouse sera). FIG. 43C: HI stalk IgG2a antibodies (D84) (pooled sera). FIG 43D: HI stalk IgG2a antibodies (D84) (individual mouse sera). FIG 43E: HI stalk IgG ratios (D84) (pooled sera). FIG. 43F: HI stalk IgG ratios (D84) (individual mouse sera).

[00141] FIG. 44: H3 cross-reactive antibodies.

5. DETAILED DESCRIPTION

5.1 CHIMERIC INFLUENZA VIRUS HEMAGGLUTININ POLYPEPTIDES

[00142] Provided herein are chimeric influenza virus hemagglutinin polypeptides comprising or consisting of an influenza virus hemagglutinin head domain polypeptide and an influenza virus hemagglutinin stem domain polypeptide, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide (e.g., the influenza virus hemagglutinin head domain polypeptide and the influenza virus hemagglutinin stem domain polypeptide are derived from different influenza virus hemagglutinin subtypes). Influenza virus hemagglutinin head domain polypeptides are described in Section 5.2 below, as well as in International Publication Nos. WO 2010/117786, WO

2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety. Influenza virus hemagglutinin stem domain polypeptides are described in Section 5.3 below, as well as in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety.

[00143] A full-length influenza hemagglutinin typically comprises an HAl domain and an HA2 domain. The stem domain is formed by two segments of the HAl domain and most or all of the HA2 domain (e.g., the entire HA2 domain, or the portion of the HA2 domain that does not include the transmembrane and cytoplasmic domains). The two segments of the HAl 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. 1 for influenza A viruses). In certain embodiments, the chimeric influenza virus hemagglutinin polypeptides described herein maintain such a structure. That is, in certain embodiments, the chimeric influenza virus hemagglutinin polypeptides described herein comprise a stable stem structure composed of an HAl domain and an HA2 domain, and a globular head domain separating the two segments of the HAl domain (in primary sequence), wherein said globular head domain is heterologous to the stem domain formed by the other segments of the HAl domain and the HA2 domain.

[00144] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises an influenza virus hemagglutinin stem domain polypeptide and an influenza virus hemagglutinin head domain polypeptide, wherein the influenza virus

hemagglutinin head domain polypeptide is heterologous to the influenza virus hemagglutinin stem domain polypeptide, and wherein the chimeric influenza virus hemagglutinin polypeptide has a primary structure of, in the following order: an HAl N-terminal stem segment, an influenza virus hemagglutinin head domain polypeptide, an HAl C-terminal stem segment and an HA2. In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises an influenza virus hemagglutinin stem domain polypeptide and an influenza virus hemagglutinin head domain polypeptide, wherein the influenza virus hemagglutinin head domain polypeptide is heterologous to the influenza virus hemagglutinin stem domain polypeptide, and wherein the chimeric influenza virus hemagglutinin polypeptide has a primary structure of, in the following order: an HAl N-terminal stem segment, an influenza virus hemagglutinin head domain polypeptide, an HAl C-terminal stem segment, and a portion of the HA2 domain (e.g., the HA2 domain lacking the transmembrane and cytoplasmic domains). The primary sequence of a chimeric influenza virus hemagglutinin polypeptide provided herein might be formed by a single polypeptide, or it might be formed by multiple polypeptides. Typically, a single polypeptide is expressed by any technique deemed suitable by one of skill in the art.

[00145] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein is monomeric. In certain embodiments, a chimeric influenza virus

hemagglutinin polypeptide provided herein is multimeric. In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein is trimeric.

[00146] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a signal peptide. Typically, the signal peptide is cleaved during or after polypeptide expression and translation to yield a mature chimeric influenza virus hemagglutinin polypeptide. In certain embodiments, also provided herein are mature chimeric influenza vims hemagglutinin polypeptides that lack a signal peptide. In embodiments where a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a signal peptide, the signal peptide might be based on any influenza virus signal peptide known to those of skill in the art. In certain embodiments, the signal peptides are based on influenza A signal peptides. In certain embodiments, the signal peptides are based on the signal peptide 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. In certain embodiments, the signal peptide might be any signal peptide deemed useful to one of skill in the art. In certain embodiments, the signal peptide is selected from SEQ ID NOS: 18-33 (see Table 4). In certain embodiments, the signal peptide is based on an influenza B virus HA signal peptide.

[00147] Table 4. Exemplary signal peptide sequences.

Description Sequence

Exemplary influenza A HA subtype HI signal MKANLLVLLCALAAADA (SEQ ID peptide NO: 18)

Exemplary influenza A HA subtype H2 signal M All YLILLF T A VRG (SEQ ID NO: 19) peptide

Exemplary influenza A HA subtype H3 signal MKTIIALSYIFCLALG (SEQ ID NO:20) peptide

Exemplary influenza A HA subtype H4 signal MLSIVILFLLIAENSS (SEQ ID NO:21) peptide

Exemplary influenza A HA subtype H5 signal MLSIVILFLLIAENSS (SEQ ID NO:22) peptide

Exemplary influenza A HA subtype H6 signal MIAIIVVAILATAGRS (SEQ ID NO:23) peptide

Exemplary influenza A HA subtype H7 signal MNTQILVF ALVA VIP TNA (SEQ ID peptide NO:24)

Exemplary influenza A HA subtype H8 signal MEKFIAIATLASTNAY (SEQ ID NO:25) peptide

Exemplary influenza A HA subtype H9 signal METKAIIAALLMVTAA (SEQ ID NO:26) peptide

Exemplary influenza A HA subtype H10 signal MYKVVVIIALLGAVKG (SEQ ID NO:27) peptide

Exemplary influenza A HA subtype HI 1 signal MEKTLLFAAIFLCVKA (SEQ ID NO:28) peptide

Exemplary influenza A HA subtype H12 signal MEKFIILSTVLAASFAY (SEQ ID NO:29) peptide

Exemplary influenza A HA subtype HI 3 signal MALNVIATLTLISVCVHA (SEQ ID peptide NO:30)

Exemplary influenza A HA subtype H14 signal MIALILVALALSHTAYS (SEQ ID NO:31) peptide Description Sequence

Exemplary influenza A HA subtype HI 5 signal MNTQIIVILVLGLSMVKS (SEQ ID peptide NO:32)

Exemplary influenza A HA subtype HI 6 signal MMIKVLYFLIIVLGRYSKA (SEQ ID peptide NO:33)

[00148] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a luminal domain. In embodiments where a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a luminal domain, the luminal domain might be based on any influenza luminal domain known to those of skill in the art. In certain embodiments, the luminal domains are based on influenza A luminal domains. In certain embodiments, the luminal domains are based on the luminal 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. In certain embodiments, the luminal domain is based on an influenza B virus luminal domain. In certain embodiments, the luminal domain might be any luminal domain deemed useful to one of skill in the art. In certain embodiments, the luminal domain is selected from SEQ ID NOS:51-66 (see Table 5). In certain embodiments, the luminal domains are from the same hemagglutinin as the stem domain. In certain embodiments, the luminal domains are from influenza virus strain or subtype as the stem domain HA2 subunit.

[00149] Table 5. Exemplary influenza virus luminal domain sequences.

Description Sequence

domain

Exemplary influenza A HA2 domain subtype H14 Luminal MGYKD (SEQ ID NO: 64) domain

Exemplary influenza A HA2 domain subtype HI 5 Luminal SGYKD (SEQ ID NO: 65) domain

Exemplary influenza A HA2 domain subtype HI 6 Luminal DNVYK (SEQ ID NO: 66) domain

[00150] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a transmembrane domain. In embodiments where a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a transmembrane domain, the transmembrane domain might be based on any influenza virus transmembrane domain known to those of skill in the art. In certain embodiments, the transmembrane domains are based on influenza A virus transmembrane domains. In specific embodiments, the transmembrane domains are based on influenza virus HA transmembrane domains (e.g., influenza A virus HA transmembrane domains or influenza B virus HA transmembrane domains). In certain embodiments, the transmembrane domains are based on a transmembrane 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. In certain embodiments, the transmembrane domain might be any transmembrane domain deemed useful to one of skill in the art. In certain embodiments, the transmembrane domain is selected from SEQ ID NOS:67-82 (see Table 6). In certain embodiments, the transmembrane domain is based on an influenza B virus transmembrane domain. In certain embodiments, the transmembrane domains are from the same hemagglutinin as the stem domain. In certain embodiments, the transmembrane domains are from influenza virus strain or subtype as the stem domain HA2 subunit.

[00151] Table 6. Exemplary influenza virus HA transmembrane domain sequences.

Description Sequence

Exemplary influenza A HA2 domain subtype ILSIYSTVASSLALAIMIAGLSFWMCS H5 Transmembrane domain (SEQ ID NO:71)

Exemplary influenza A HA2 domain subtype ILAIYSTVSSSLVLVGLIIAVGLWMCS H6 Transmembrane domain (SEQ ID NO:72)

Exemplary influenza A HA2 domain subtype VILWF SFGASCFLLL AIAMGLVFICVK H7 Transmembrane domain (SEQ ID NO:73)

Exemplary influenza A HA2 domain subtype ILSIYSTVAASLCLAILIAGGLILGMQ H8 Transmembrane domain (SEQ ID NO:74)

Exemplary influenza A HA2 domain subtype ILTIYSTVAS SLVL AMGF AAFLF W AMS H9 Transmembrane domain (SEQ ID NO:75)

Exemplary influenza A HA2 domain subtype IILWF SFGESCF VLL AVVMGLVFFCLK H10 Transmembrane domain (SEQ ID NO:76)

Exemplary influenza A HA2 domain subtype IL SI YS CI A S SL VL A ALIMGFMF W AC S Hl l Transmembrane domain (SEQ ID NO:77)

Exemplary influenza A HA2 domain subtype ILSIYSSVASSLVLLLMIIGGFIFGCQN HI 2 Transmembrane domain (SEQ ID NO:78)

Exemplary influenza A HA2 domain subtype ALSIYSCIASSVVLVGLILSFIMWACSS HI 3 Transmembrane domain (SEQ ID NO:79)

Exemplary influenza A HA2 domain subtype IILWISF SMSCF VF VALILGF VLWACQ HI 4 Transmembrane domain (SEQ ID NO:80)

Exemplary influenza A HA2 domain subtype VILWF SFGASCVMLL AIAMGLIFMC VKN HI 5 Transmembrane domain (SEQ ID NO:81)

Exemplary influenza A HA2 domain subtype VL SIYSCI AS SIVLVGLILAFIMWAC S HI 6 Transmembrane domain (SEQ ID NO: 82)

[00152] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a cytoplasmic domain. In embodiments where a chimeric influenza virus hemagglutinin polypeptide provided herein comprises a cytoplasmic domain, the cytoplasmic domain might be based on any influenza cytoplasmic domain known to those of skill in the art. In specific embodiments, the transmembrane domains are based on influenza virus HA cytoplasmic domains (e.g., influenza A virus HA cytoplasmic domains or influenza B virus HA cytoplasmic domains). In certain embodiments, the cytoplasmic domains are based on influenza A virus cytoplasmic domains. In certain embodiments, the cytoplasmic domains are based on a cytoplasmic 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 HI 8. In certain embodiments, the cytoplasmic domain is based on a cytoplasmic domain of an influenza B virus HA. In certain embodiments, the cytoplasmic domain might be any cytoplasmic domain deemed useful to one of skill in the art. In certain embodiments, the cytoplasmic domain is selected from SEQ ID NOS:83-98 (see Table 7). In certain embodiments, the cytoplasmic domains are from the same hemagglutinin as the stem domain. In certain embodiments, the cytoplasmic domains are from influenza virus strain or subtype as the stem domain HA2 subunit.

[00153] Table 7. Exemplary influenza virus HA cytoplasmic domain sequences.

[00154] In certain embodiments, the chimeric influenza virus hemagglutinin polypeptides provided herein further comprise one or more polypeptide domains. Useful polypeptide domains include domains that facilitate purification, folding and cleavage of portions of a polypeptide. For example, a His tag (His-His-His-His-His-His, SEQ ID NO: 101), FLAG epitope or other purification tag can facilitate purification of a chimeric influenza virus hemagglutinin

polypeptide provided herein. In some embodiments, 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. In some embodiments, the chimeric influenza virus hemagglutinin polypeptides provided herein comprise a foldon, or trimenzation domain. In specific embodiments, the chimeric influenza vims hemagglutinin polypeptides provided herein comprise a foldon, or trimerization, domain from bacteriophage T4 fibritin. A foldon, or trimerization, domain from bacteriophage T4 fibritin can facilitate trimerization of polypeptides provided herein. In some embodiments, 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, PLoSONE 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.

Chem. 279(10):8991-8998, the contents of which are hereby incorporated by reference in their entirety. Examples include GSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 102). A foldon domain can be useful to facilitate trimerization of soluble polypeptides provided herein. In specific embodiments, the chimeric influenza vims hemagglutinin polypeptides provided herein comprise a cleavage site. 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). In certain embodiments, the cleavage site is a cleavage site recognized by Tobacco Etch Vims (TEV) protease {e.g., amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln- (Gly/Ser) (SEQ ID NO:50).

[00155] In certain embodiments, the chimeric influenza hemagglutinin polypeptides are soluble polypeptides, such as those described in Examples 6 and 9 of International Publication No. WO 2013/043729 and U.S. Patent Application Publication No. 2015/0132330, each of which is incorporated herein by reference in its entirety. In some embodiments, a chimeric influenza hemagglutinin polypeptide is a soluble polypeptide, which comprises a foldon or trimerization domain (e.g., a foldon, or trimerization, domain from bacteriophage T4 fibritin).

[00156] In certain embodiments, the chimeric influenza vims hemagglutinin polypeptides provided herein are capable of forming a three dimensional stmcture that is similar to the three dimensional structure 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 a chimeric influenza virus hemagglutinin 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 [DOI: 10.1126/science. l l71491], and Kashyap et al, 2008, Proc. Natl. Acad. Sci. USA 105(16):5986-5991, the contents of which are hereby incorporated by reference in their entireties. In certain embodiments, 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.

[00157] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide described herein retains one, two, or more, or all of the functions of a wild-type influenza virus HA. Nonlimiting examples of functions of a wild-type influenza virus HA include fusogenic activity, receptor binding activity, budding, and particle formation. In a specific embodiment, a chimeric influenza hemagglutinin (HA) polypeptide described herein has fusogenic activity. Assays known to one skilled in the art can be utilized the assess the fusogenic activity of a chimeric influenza hemagglutinin (HA) polypeptide described herein, such as, for example, immunofluorescence assays and pseudotyped virus-like-particle assays.

[00158] In certain embodiments, 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). In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide described herein may be conjugated to immunomodulatory molecules, such as proteins which would target the chimeric influenza hemagglutinin (HA) polypeptide to immune cells such as B cells {e.g., C3d) or T cells. In certain embodiments, 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).

[00159] Also provided herein are chimeric influenza hemagglutinin polypeptides comprising an HA2 subunit and a chimeric HAl subunit. In certain embodiments, the chimeric HAl subunit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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. In certain embodiments, the chimeric 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 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. In certain embodiments, 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 HAl domain. In specific embodiments, the chimeric HAl subunit comprises an influenza virus hemagglutinin head domain polypeptide comprising amino acids of two or more different subtypes or strains of influenza virus. In specific embodiments, the chimeric HAl subunit comprises a globular head with amino acids of two or more different subtypes or strains of influenza virus.

[00160] It will be understood by those of skill in the art that the chimeric influenza virus hemagglutinin polypeptides provided herein can be prepared according to any technique known by and deemed suitable to those of skill in the art, including the techniques described herein. In certain embodiments, the chimeric influenza virus hemagglutinin polypeptides are isolated.

5.1.1 A/A, A B, AND B B CHIMERIC INFLUENZA VIRUS HA POLYPEPTIDES

[00161] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide described herein comprises or consists of (i) an influenza virus hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S.

Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875, and 2018-0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety, or an influenza virus hemagglutinin stem domain polypeptide from any known strain or subtype of influenza virus (e.g., any wild- type influenza virus hemagglutinin stem domain polypeptide such as the stem domain of the hemagglutinin of an influenza virus described in Section 5.6 below) and (ii) an influenza virus hemagglutinin head domain polypeptide described herein (see, e.g., Section 5.2 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875, and 2018- 0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety, or an influenza virus hemagglutinin head domain polypeptide from any known strain or subtype of influenza virus (e.g., any wild-type influenza virus hemagglutinin head domain polypeptide), wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide. In specific embodiments, the influenza virus hemagglutinin head domain polypeptide is not an influenza virus hemagglutinin head domain polypeptide of influenza A virus subtype HI or H3. In some embodiments, the influenza virus hemagglutinin head domain polypeptide is not an influenza virus hemagglutinin head domain polypeptide of influenza A virus subtype H2. In certain embodiments, the influenza virus hemagglutinin head domain polypeptide is not an influenza virus hemagglutinin head domain polypeptide of influenza A virus subtype H5. In such embodiments, an influenza hemagglutinin head domain polypeptide (which is heterologous to the influenza hemagglutinin stem domain polypeptide) is located, in primary sequence, between the HA1 N-terminal stem segment and HA1 C-terminal stem segment of the influenza hemagglutinin stem domain.

[00162] In certain embodiments, the influenza hemagglutinin stem domain polypeptides of the chimeric influenza virus hemagglutinin polypeptides described herein maintain the cysteine residues identified in influenza hemagglutinin polypeptides as Ap and Aq in Fig. 1, i.e., the cysteine residues identified in influenza hemagglutinin polypeptides as Ap and Aq in Fig. 1 are maintained in the chimeric influenza virus hemagglutinin polypeptides described herein. Thus, in certain embodiments, in the primary sequence of a chimeric influenza virus hemagglutinin polypeptide described herein: (i) the N-terminal segment of an influenza hemagglutinin stem domain polypeptide ends at the cysteine residue identified as Ap in Fig. 1, (ii) the C-terminal segment of an influenza hemagglutinin stem domain polypeptide begins at the cysteine residue identified as A_q in Fig. 1; and (iii) the influenza hemagglutinin head domain polypeptide (which is heterologous to the influenza hemagglutinin stem domain polypeptide) is between the N- terminal and C-terminal stem segments of the influenza hemagglutinin stem domain polypeptide. In such embodiments, an influenza hemagglutinin head domain polypeptide (which is heterologous to the influenza hemagglutinin stem domain polypeptide) is located, in primary sequence, between the N-terminal and C-terminal stem segments of the influenza hemagglutinin stem domain polypeptide. In certain embodiments, the chimeric influenza virus HA polypeptide further includes an HA2 domain or a fragment thereof (e.g., a portion of the HA2 domain lacking the transmembrane and cytoplasmic domains).

[00163] In certain embodiments, the influenza B virus hemagglutinin stem domain

polypeptides of the chimeric influenza virus hemagglutinin polypeptides described herein maintain the alanine residues identified in influenza hemagglutinin polypeptides as Dp and Dq.

Thus, in certain embodiments, in the primary sequence of a chimeric influenza virus

hemagglutinin polypeptide described herein: (i) the HA1 N-terminal stem segment of an influenza B virus hemagglutinin stem domain polypeptide ends at the alanine residue

corresponding to Dp, (ii) the HA1 C-terminal stem segment of an influenza B virus

hemagglutinin stem domain polypeptide begins at the alanine residue corresponding to Dq; and

(iii) the influenza A virus hemagglutinin head domain polypeptide is between the HA1 N- terminal stem segment and HA1 C-terminal stem segments of the influenza B virus

hemagglutinin stem domain polypeptide. In certain embodiments, the chimeric influenza virus HA polypeptide further includes an HA2 domain or a portion thereof (e.g., a portion of the HA2 domain lacking the transmembrane and cytoplasmic domains). Influenza B virus hemagglutinin stem domain polypeptides are described in detail in Section 5.3 below.

[00164] In a specific embodiment, provided herein is a chimeric influenza virus

hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S.

Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018-0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 (which are incorporated herein by reference in their entirety), or an influenza virus

hemagglutinin stem domain polypeptide from any known strain or subtype of influenza virus (e.g., any wild-type influenza virus hemagglutinin stem domain polypeptide) and (ii) an influenza vims hemagglutinin head domain polypeptide from influenza A vims subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hl l, H12, H13, H14, H15, H16, H17, or H18, wherein said influenza vims hemagglutinin head domain polypeptide is heterologous to said influenza vims hemagglutinin stem domain polypeptide.

[00165] In another specific embodiment, provided herein is a chimeric influenza vims hemagglutinin polypeptide comprising or consisting of (i) an influenza vims hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018-0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 (which are incorporated herein by reference in their entirety), or an influenza vims

hemagglutinin stem domain polypeptide from any known strain or subtype of influenza vims (e.g., any wild-type influenza vims hemagglutinin stem domain polypeptide) and (ii) an influenza vims hemagglutinin head domain polypeptide from influenza A vims subtype H4, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18, wherein said influenza vims hemagglutinin head domain polypeptide is heterologous to said influenza vims hemagglutinin stem domain polypeptide.

[00166] In another specific embodiment, provided herein is a chimeric influenza vims hemagglutinin polypeptide comprising or consisting of (i) an influenza vims hemagglutinin stem domain polypeptide from influenza A vims subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, HI 2, HI 3, HI 4, HI 5, HI 6, HI 7, or HI 8; and (ii) an influenza vims hemagglutinin head domain polypeptide described herein (see, e.g., Section 5.2 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018-0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety, or an influenza vims hemagglutinin head domain polypeptide from any known strain or subtype of influenza vims (e.g., any wild- type influenza vims hemagglutinin stem domain polypeptide), wherein said influenza vims hemagglutinin head domain polypeptide is heterologous to said influenza vims hemagglutinin stem domain polypeptide. [00167] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, HI 1, HI 2, HI 3, HI 4, HI 5, HI 6, HI 7, or HI 8; and (ii) an influenza virus hemagglutinin head domain polypeptide from influenza A virus subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, 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.

[00168] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018-0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 which are incorporated herein by reference in their entirety, or an influenza virus hemagglutinin stem domain polypeptide from any known strain or subtype of influenza virus (e.g., any wild- type influenza virus hemagglutinin stem domain polypeptide) and (ii) an influenza virus hemagglutinin head domain polypeptide from avian influenza virus subtype HI, H2, or H3, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide.

[00169] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018-0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 which are incorporated herein by reference in their entirety, or an influenza virus hemagglutinin stem domain polypeptide from any known strain or subtype of influenza virus (e.g., any wild- type influenza virus hemagglutinin stem domain polypeptide) and (ii) an influenza virus hemagglutinin head domain polypeptide from horse influenza virus subtype H3, wherein said influenza vims hemagglutinin head domain polypeptide is heterologous to said influenza vims hemagglutinin stem domain polypeptide.

[00170] In another specific embodiment, provided herein is a chimeric influenza vims hemagglutinin polypeptide comprising or consisting of (i) an influenza vims hemagglutinin stem domain polypeptide from an influenza A vims of subtype HI and (ii) an influenza vims hemagglutinin head domain polypeptide from an influenza A vims 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 vims hemagglutinin head domain polypeptide is heterologous to said influenza vims

hemagglutinin stem domain polypeptide. In a specific embodiment, the influenza vims hemagglutinin head domain polypeptide is from an influenza A vims of subtype H5, H6, H8, H9, HI 1, H12, H13, H16, H17, or H18. In a specific embodiment, the influenza vims hemagglutinin head domain polypeptide is from an avian influenza A vims of subtype H5, H6, H8, H9, HI 1, H12, H13, H16, H17, or H18. In another specific embodiment, the influenza vims

hemagglutinin head domain polypeptide is not from an influenza A vims of subtype HI, H2, or H3. In another specific embodiment, the influenza vims hemagglutinin head domain polypeptide is not from an influenza A vims of subtype H5.

[00171] In another specific embodiment, provided herein is a chimeric influenza vims hemagglutinin polypeptide comprising or consisting of (i) an influenza vims hemagglutinin stem domain polypeptide from an influenza A vims of subtype H3 and (ii) an influenza vims hemagglutinin head domain polypeptide from an influenza A vims 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 vims hemagglutinin head domain polypeptide is heterologous to said influenza vims

hemagglutinin stem domain polypeptide. In a specific embodiment, the influenza vims hemagglutinin head domain polypeptide is from an influenza A vims of subtype H4, H7, H10, H14, or H15. In a specific embodiment, the influenza vims hemagglutinin head domain polypeptide is from an avian influenza A vims of subtype H4, H7, H10, H14, or H15. In another specific embodiment, the influenza vims hemagglutinin head domain polypeptide is not from an influenza A vims of subtype HI, H2, or H3. In another specific embodiment, the influenza vims hemagglutinin head domain polypeptide is not from an influenza A vims of subtype H5.

[00172] In another specific embodiment, provided herein is a chimeric influenza vims hemagglutinin polypeptide comprising or consisting of (i) an influenza vims hemagglutinin stem domain polypeptide from an influenza A virus of subtype 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, HIO, 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. In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from an influenza A virus of subtype H2 and (ii) an influenza virus hemagglutinin head domain polypeptide from an avian influenza A virus of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hl l, H12, H13, H14, H15, H16, H17, or HI 8, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide. In a specific embodiment, the 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.

[00173] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from an influenza A virus of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hl l, H12, H13, H14, H15, H16, H17, or H18 and (ii) an influenza virus hemagglutinin head domain polypeptide from an influenza B virus, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide.

[00174] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide described herein comprises or consists of (i) an influenza B virus hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S.

Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018-0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety, or an influenza B virus

hemagglutinin stem domain polypeptide from any known strain or lineage of influenza B virus (e.g., any wild-type influenza B virus hemagglutinin stem domain polypeptide such as the stem domain of the hemagglutinin of an influenza B virus described in Section 5.6 below) and (ii) an influenza A virus hemagglutinin head domain polypeptide described herein (see, e.g., Section 5.2 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO

2013/043729, and WO 2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875, and 2018-0008696and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety, or an influenza A virus hemagglutinin head domain polypeptide from any known strain or subtype of influenza A virus (e.g., any wild-type influenza A virus hemagglutinin head domain polypeptide). In specific embodiments, the influenza A virus hemagglutinin head domain polypeptide is not an influenza virus hemagglutinin head domain polypeptide of influenza A virus subtype HI or H3. In some embodiments, the influenza A virus hemagglutinin head domain polypeptide is not an influenza virus hemagglutinin head domain polypeptide of influenza A virus subtype H2. In certain embodiments, the influenza A virus hemagglutinin head domain polypeptide is not an influenza virus hemagglutinin head domain polypeptide of influenza A virus subtype H5.

[00175] In a specific embodiment, provided herein is a chimeric influenza virus

hemagglutinin polypeptide comprising or consisting of (i) an influenza B virus hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO

2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018- 0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330, which are incorporated herein by reference in their entirety, or an influenza B virus hemagglutinin stem domain polypeptide from any known strain or lineage of influenza B virus (e.g., any wild-type influenza B virus hemagglutinin stem domain polypeptide) and (ii) an influenza A virus hemagglutinin head domain polypeptide from influenza A virus subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18.

[00176] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza B virus hemagglutinin stem domain polypeptide described herein (see, e.g., Section 5.3 below) or in International Publication Nos. WO 2010/117786, WO 2011/123495, WO 2013/043729, and WO

2014/099931, U.S. Publication Nos. 2010/0297174, 2013/0129761, 2014/0328875 and 2018- 0008696, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 which are incorporated herein by reference in their entirety, or an influenza B virus hemagglutinin stem domain polypeptide from any known strain or lineage of influenza B virus (e.g., any wild-type influenza B virus hemagglutinin stem domain polypeptide) and (ii) an influenza A virus hemagglutinin head domain polypeptide from influenza A virus subtype H4, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18.

[00177] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza B virus hemagglutinin stem domain polypeptide from influenza B virus Yamagata lineage; and (ii) an influenza virus hemagglutinin head domain polypeptide from influenza A virus 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.

[00178] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza B virus hemagglutinin stem domain polypeptide from influenza B virus Victoria lineage; and (ii) an influenza virus hemagglutinin head domain polypeptide from influenza A virus 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.

[00179] In certain embodiments, a chimeric influenza virus HA polypeptide is a polypeptide described in Ermler et al., 2017, "Chimeric Hemagglutinin Constructs Induce Broad Protection Against Influenza B Virus Challenge in the Mouse Model," Journal of Virology, 91(12), e00286- 17, which is incorporated herein by reference in its entirety.

[00180] In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises an influenza A virus hemagglutinin stem domain polypeptide and an influenza A virus hemagglutinin head domain polypeptide, wherein the chimeric influenza virus hemagglutinin polypeptide has a primary structure of, in the following order: an HA1 N-terminal stem segment, an influenza virus hemagglutinin head domain polypeptide, an HA1 C-terminal stem segment and an HA2. In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises an influenza B virus hemagglutinin stem domain polypeptide and an influenza A virus hemagglutinin head domain polypeptide, wherein the chimeric influenza virus hemagglutinin polypeptide has a primary structure of, in the following order: an HAl N-terminal stem segment, an influenza virus hemagglutinin head domain polypeptide, an HAl C-terminal stem segment and an HA2. In certain embodiments, a chimeric influenza virus hemagglutinin polypeptide provided herein comprises an influenza virus hemagglutinin stem domain polypeptide (e.g,. an influenza B virus hemagglutinin stem domain polypeptide or influenza A virus hemagglutinin stem domain polypeptide) and an influenza virus hemagglutinin head domain polypeptide (e.g,. an influenza A virus hemagglutinin stem domain polypeptide), wherein the influenza virus hemagglutinin head domain polypeptide is

heterologous to the influenza virus hemagglutinin stem domain polypeptide, and wherein the chimeric influenza virus hemagglutinin polypeptide has a primary structure of, in the following order: an HAl N-terminal stem segment, an influenza virus hemagglutinin head domain polypeptide, an HAl C-terminal stem segment, and a portion of the HA2 domain (e.g., the HA2 domain lacking the transmembrane and cytoplasmic domains). The primary sequence of a chimeric influenza virus hemagglutinin polypeptide provided herein might be formed by a single polypeptide, or it might be formed by multiple polypeptides. Typically, a single polypeptide is expressed by any technique deemed suitable by one of skill in the art.

[00181] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from an influenza B virus 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian influenza A virus of subtype H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18. In another specific embodiment, the 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. [00182] In another specific embodiment provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from an influenza B virus and (ii) an influenza virus hemagglutinin head domain polypeptide from an influenza B virus, wherein said influenza virus hemagglutinin head domain polypeptide is heterologous to said influenza virus hemagglutinin stem domain polypeptide.

[00183] In a specific embodiment, the chimeric influenza virus HA polypeptide is a chimeric influenza virus HA polypeptide described in Section 6 below (e.g., in Sections 6.1 to 6.5 below) or in Section 5.1.1.1, 5.1.1.2, or 5.1.1.3.

5.1.1.1 A/A Chimeric HA Polypeptides

[00184] In a specific embodiment, provided herein is a chimeric influenza virus

hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/California/7/2009 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5, H6, H8, H9, HI 1, H12, H13, or H16. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian influenza A virus of subtype H5, H6, H8, H9, HI 1, H12, H13, or H16. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00185] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/California/7/2009 (HI) and (ii) an influenza virus hemagglutinin head domain polypeptide from an influenza A virus of subtype H2, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian influenza A virus of subtype H2, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H6. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H7. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H8. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H9. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H10. In a specific

embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype HI 1. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H12. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype HI 3. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H14. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H15. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype HI 6.

[00186] In another specific embodiment, provided herein is 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5, H6, H8, H9, HI 1, H12, H13, or H16. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00187] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/South Carolina/1918 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5, H6, H8, H9, HI 1, H12, H13, or H16. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00188] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/US SR/92/ 1977 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5, H6, H8, H9, HI 1, H12, H13, or H16. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00189] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/California/04/2009 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5, H6, H8, H9, HI 1, H12, H13, or H16. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H2 (e.g., A/Singapore/ 1/57), subtype H4 (e.g., A/duck/Czech/56), subtype H5 (e.g.,

A/Vietnam/1203/04), subtype H6 (e.g., A/mallard/Sweden/81/02), subtype H8 (e.g.,

A/mallard/Sweden/24/02), subtype H9 (e.g., A/guinea fowl/Hong Kong/WF 10/99), subtype HI 1 (e.g., A/Northern shovel er/Netherlands/18/99), subtype H12 (e.g., A/mallard/Interior

Alaska/7MP0167/07), subtype H13 (e.g., A/black headed gull/Swedend/1/99), or subtype H16 (e.g., A/black headed gull/Swedend/5/99). In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00190] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/Perth/16/2009 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4, H7, H10, H14, or H15. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from A/Viet Nam/1203/04 (H5). In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4 (e.g. A/duck/Czech/56), subtype H10 (e.g., A/mallard/Interior or Alaska/10BM01929/10), subtype H14 (e.g.,

A/mallard/Gurjev/263/82), or subtype H15 (e.g., A/wedge tailed shearwater/Western

Australia/2576/79). In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H7 (e.g., A/mallard/ Alberta/24/01 or A/Anhui/1/13). In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from A/Alberta/24/01 (H7). In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5. [00191] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/Brisbane/lO/2007-like (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, HIO, 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian influenza A virus of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hl l, H12, H13, H14, H15, H16, H17, or H18. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4, H7, HIO, H14, or H15. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00192] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/Hong Kong/1/1968 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4, H7, H10, H14, or H15. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00193] In another specific embodiment, provided herein is 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, HIO, 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian influenza A virus of subtype HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hl l, H12, H13, H14, H15, H16, H17, or H18. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4, H7, HIO, H14, or H15. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00194] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/Hong Kong/4801/14 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4, H7, H10, H14, or H15. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4 (e.g.

A/duck/Czech/56), subtype H5 (e.g., A/Viet Nam/1203/04), subtype H7 (e.g.,

A/mallard/ Alberta/24/01 or A/Anhui/1/13), subtype HIO (e.g., A/mallard/Interior or

Alaska/10BM01929/10), subtype H14 (e.g., A/mallard/Gurjev/263/82), or subtype H15 (e.g., A/wedge tailed shearwater/Western Australia/2576/79).

[00195] In another specific embodiment, provided herein is 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

hemagglutinin stem domain polypeptide. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H4, H7, H10, H14, or H15. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5.

[00196] In another specific embodiment, provided herein is a chimeric influenza virus hemagglutinin polypeptide comprising or consisting of (i) an influenza virus hemagglutinin stem domain polypeptide from influenza A virus A/Puerto Rico/8/1934 (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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an avian 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. In a specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype HI, H2, H4, H5, H6, H7, H9, H10, H14, or H15. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype HI, H2, H5, H6, or H9. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype HI . In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H2. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H3. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is not from an influenza A virus of subtype H5. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H5. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from A/VietNam/1203/04 (H5). In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from an influenza A virus of subtype H6. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from

A/mallard/Sweden/81/02 (H6). In another specific embodiment, the influenza virus

hemagglutinin head domain polypeptide is from an influenza A virus of subtype H9. In another specific embodiment, the influenza virus hemagglutinin head domain polypeptide is from A/guinea fowl/Hong Kong/WF 10/99 (H9).

[00197] Table 8. Exemplary cH2/l, cH4/l, cH5/l, cH6/l, cH8/l, cH9/l, cHl 1/1, cH12/l, CH13/1, cH16/l, cH4/3, cH5/3, cH7/3, cHlO/3, cH14/3, cH15/3 chimeric influenza

hemagglutinin polypeptides.

Chimeric Strain of Influenza Virus HA Globular Strain of Influenza Virus influenza Head Domain HA Stem Domain

hemagglutinin

polypeptide

cH4/l A/duck/Czech/56 (H4) (or the globular head A/California/04/2009 (HI) (or domain of an A/duck/Czech/56-like the stem domain of an influenza virus) A/California/04/2009 cH5/l Any H5 A/California/4/2009 (HI) (or

A/California/4/2009-like) cH5/l A/Vietnam/1203/2004 (H5) (or the globular A/California/4/2009 (HI) (or head domain of an A/Vietnam/1203/2004- A/California/4/2009-like) like influenza virus)

cH5/l A/Indonesia/5/2005 (H5) A/California/4/2009 (HI) (or

A/California/4/2009-like) cH5/l A/Anhui/1/2005 (H5) A/California/4/2009 (HI) (or

A/California/4/2009-like) cH5/l A/bar headed goose/Quinghai/lA/2005 (H5) A/California/4/2009 (HI) (or

A/California/4/2009-like) cH5/l A/turkey/Turkey/1/2005 (H5) A/California/4/2009 (HI) (or

A/California/4/2009-like) cH5/l A/whooperswan/Mongolia/244/2005 (H5) A/California/4/2009 (HI) (or

A/California/4/2009-like) cH5/l Any H5 A/PR/8/34 (HI) (or the stem domain of an A/PR/8/34-like influenza virus)

cH5/l A/Vietnam/1203/2004 (H5) (or the globular A/PR/8/34 (HI) (or the stem head domain of an A/Vietnam/1203/2004- domain of an A/PR/8/34-like like influenza virus) influenza virus)

cH6/l Any H6 A/California/04/2009 (HI) (or the stem domain of an A/California/04/2009-like influenza virus)

cH6/l A/mallard/Sweden/81/02 (H6) (or the A/California/04/2009 (HI) (or globular head domain of an the stem domain of an A/mallard/Sweden/81/02-like influenza A/California/04/2009-like virus) influenza virus)

cH6/l Any H6 A/PR/8/34 (HI) (or the stem domain of an A/PR/8/34-like influenza virus)

cH6/l A/mallard/Sweden/81/02 (H6) (or the A/PR/8/34 (HI) (or the stem globular head domain of an domain of an A/PR/8/34-like A/mallard/Sweden/81/02-like influenza influenza virus)

virus)

cH8/l Any H8 A/California/04/2009 (HI) (or the stem domain of an A/California/04/2009-like Chimeric Strain of Influenza Virus HA Globular Strain of Influenza Virus influenza Head Domain HA Stem Domain

hemagglutinin

polypeptide

influenza virus)

cH8/l A/mallard/Sweden/24/02 (H8) (or the A/California/04/2009 (HI) (or globular head domain of an the stem domain of an

A/mallard/Sweden/24/02-like influenza A/California/04/2009-like virus) influenza virus)

cH9/l Any H9 A/PR/8/34 (HI) (or the stem domain of an A/PR/8/34-like influenza virus)

cH9/l A/guinea fowl/Hong Kong/WF 10/99 (H9) A/PR/8/34 (HI) (or the stem

(or the globular head domain of an A/guinea domain of an A/PR/8/34-like fowl/Hong Kong/WF 10/99-like influenza influenza virus)

virus)

cHl l/1 Any HI 1 A California/04/2009 (HI) (or the stem domain of an A California/04/2009-like influenza virus)

cHl l/1 A/Northern shoveler/Netherlands/18/1999 A California/04/2009 (HI) (or

(HI 1) (or the globular head domain of an the stem domain of an

A/Northern shoveler/Netherlands/18/1999- A California/04/2009-like like influenza virus) influenza virus)

CH12/1 Any H12 A California/04/2009 (HI) (or the stem domain of an A California/04/2009-like influenza virus)

CH12/1 A/mallard/Interior Alaska/7MP0167/2007 A California/04/2009 (HI) (or

(H12) (or the globular head domain of an the stem domain of an

A/mallard/Interior Alaska/7MP0167/2007- A California/04/2009-like like influenza virus) influenza virus)

CH13/1 Any HI 3 A California/04/2009 (HI) (or the stem domain of an A California/04/2009-like influenza virus)

CH13/1 A/black headed gull/Sweden/ 1/99 (HI 3) (or A California/04/2009 (HI) (or the globular head domain of an A/black the stem domain of an headed gull/Sweden/l/99-like influenza A California/04/2009-like virus) influenza virus)

CH16/1 Any HI 6 A California/04/2009 (HI) (or the stem domain of an A California/04/2009-like influenza virus)

CH16/1 A/black headed gull/Sweden/5/99 (HI 6) (or A California/04/2009 (HI) (or the globular head domain of an A/black the stem domain of an

Chimeric Strain of Influenza Virus HA Globular Strain of Influenza Virus influenza Head Domain HA Stem Domain hemagglutinin

polypeptide

cH7/3 A/chicken/Jalisco/CPAl/2012 (H7) A/Indiana/10/2011 (H3) cH7/3 A/mallard/Alberta/24/2001 (H7) A/Indiana/10/2011 (H3) cH7/3 A/rhea/NC/39482/93 (H7) A/Indiana/10/2011 (H3) cH7/3 A/mallard/Netherlands/12/2000 (H7) A/Indiana/10/2011 (H3) cH7/3 Any H7 A/Perth/16/2009 (H3) cH7/3 A/Netherlands/219/03 (H7) A/Perth/16/2009 (H3) cH7/3 A/Canada/504/04 (H7) A/Perth/16/2009 (H3) cH7/3 A/Canada/444/04 (H7) A/Perth/16/2009 (H3) cH7/3 A/chicken/Jalisco/CPAl/2012 (H7) A/Perth/16/2009 (H3) cH7/3 A/mallard/Alberta/24/2001 (H7) A/Perth/16/2009 (H3) cH7/3 A/rhea/NC/39482/93 (H7) A/Perth/16/2009 (H3) cH7/3 A/mallard/Netherlands/12/2000 (H7) A/Perth/16/2009 (H3) cH7/3 A/mallard/Alberta/24/2001 (H7) (or the A/Perth/16/09 (H3) (or the globular head domain of an stem domain of an

A/mallard/Alberta/24/2001 -like influenza A/Perth/16/09-like influenza virus) virus HA) cH4/3 Any H4 A/Perth/16/09 (H3) (or the stem domain of an

A/Perth/16/09-like influenza virus HA)

cH4/3 A/duck/Czech/56 (H4) (or the globular head A/Perth/16/09 (H3) (or the domain of an A/duck/Czech/56-like stem domain of an influenza virus HA) A/Perth/16/09-like influenza virus HA)

cHlO/3 Any HI 0 A/Perth/16/2009 HA (or the stem domain of an

A/Perth/16/2009-like influenza virus)

cHlO/3 A/mallard/Interior Alaska/1 OBMO 1929/10 A/Perth/16/2009 HA (or the

(H10) (or the globular head domain of an stem domain of an

A/mallard/Interior Alaska/1 OBMO 1929/10- A/Perth/16/2009-like like influenza virus) influenza virus)

CH14/3 Any H14 A/Perth/16/2009 HA (or the stem domain of an

A/Perth/16/2009-like influenza virus)

CH14/3 A/mallard/Gurj ev/263/1982 (H14) (or the A/Perth/16/2009 HA (or the globular head domain of an stem domain of an

A/mallard/Gurj ev/263/1982-like influenza A/Perth/16/2009-like virus) influenza virus)

CH15/3 Any HI 5 A/Perth/16/2009 HA (or the stem domain of an Chimeric Strain of Influenza Virus HA Globular Strain of Influenza Virus influenza Head Domain HA Stem Domain hemagglutinin

polypeptide

A/Perth/16/2009-like influenza virus)

CH15/3 A/wedge tailed shearwater/Western A/Perth/16/2009 HA (or the

Australia/2576/1979 HA (or the globular stem domain of an head domain of an A/wedge tailed A/Perth/16/2009-like shearwater/W estern Australia/2576/1979- influenza virus)

like influenza virus)

cH4/3 Any H4 A/HK/4801/14 (or the stem domain of an A/HK/4801/14- like influenza virus) cH4/3 A/duck/Czech/56 HA (or the globular head A/HK/4801/14 (or the stem domain of an A/duck/Czech/56-like domain of an A/HK/4801/14- influenza virus) like influenza virus) cH5/3 Any H5 A/HK/4801/14 (or the stem domain of an A/HK/4801/14- like influenza virus) cH5/3 A/Vietnam/1203/2004 (or the globular head A/HK/4801/14 (or the stem domain of an A/Vietnam/1203/2004-like domain of an A/HK/4801/14- influenza virus) like influenza virus) cH7/3 Any H7 A/HK/4801/14 (or the stem domain of an A/HK/4801/14- like influenza virus) cH7/3 A/Anhui/1/13 (or the globular head of an A/HK/4801/14 (or the stem

A/Anhui/l/13-like influenza virus) domain of an A/HK/4801/14- like influenza virus)

CH14/3 Any H14 A/HK/4801/14 (or the stem domain of an A/HK/4801/14- like influenza virus)

CH14/3 A/mallard/Gurjev/263/82 (or the globular A/HK/4801/14 (or the stem head of an A/mallard/Gurjev/263/82-like domain of an A/HK/4801/14- influenza virus) like influenza virus)

CH15/3 Any HI 5 A/HK/4801/14 (or the stem domain of an A/HK/4801/14- like influenza virus)

CH15/3 A/wedge tailed shearwater/Western A/HK/4801/14 (or the stem

Australia/2576/79 (or the globular head of domain of an A/HK/4801/14- an A/wedge tailed shearwater/Western like influenza virus)

Australia/2576/79)

[00198] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H5 subtype (sometimes referred to herein as a "cH5/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH5/l chimeric influenza hemagglutinin polypeptide is a cH5/l chimeric influenza hemagglutinin polypeptide described in Table 8, above, or Section 6, below.

[00199] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the H3 subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H5 subtype (sometimes referred to herein as a "cH5/3 chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH5/3 chimeric influenza hemagglutinin polypeptide is a cH5/3 chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00200] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the H3 subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H7 subtype (sometimes referred to herein as a "cH7/3 chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH7/3 chimeric influenza hemagglutinin polypeptide is a cH7/3 chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00201] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the H3 subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H4 subtype (sometimes referred to herein as a "cH4/3 chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH4/3 chimeric influenza hemagglutinin polypeptide is a cH4/3 chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00202] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the H3 subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H10 subtype (sometimes referred to herein as a "cHlO/3 chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cHlO/3 chimeric influenza hemagglutinin polypeptide is a cHlO/3 chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00203] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the H3 subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H14 subtype (sometimes referred to herein as a "cH14/3 chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH14/3 chimeric influenza hemagglutinin polypeptide is a cH14/3 chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00204] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the H3 subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the HI 5 subtype (sometimes referred to herein as a "cH15/3 chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH15/3 chimeric influenza hemagglutinin polypeptide is a cH15/3 chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00205] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H2 subtype (sometimes referred to herein as a "cH2/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH2/l chimeric influenza hemagglutinin polypeptide is a cH2/l chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00206] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H4 subtype (sometimes referred to herein as a "cH4/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH4/l chimeric influenza hemagglutinin polypeptide is a cH4/l chimeric influenza hemagglutinin polypeptide described in Table 8, above. [00207] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H6 subtype (sometimes referred to herein as a "cH6/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH6/l chimeric influenza hemagglutinin polypeptide is a cH6/l chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00208] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H8 subtype (sometimes referred to herein as a "cH8/l chimeric influenza hemagglutinin polypeptide"). "). In a specific embodiment, the cH8/l chimeric influenza hemagglutinin polypeptide is a cH8/l chimeric influenza hemagglutinin polypeptide described in Table 8, above, or Section 6, below.

[00209] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H9 subtype (sometimes referred to herein as a "cH9/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH9/l chimeric influenza hemagglutinin polypeptide is a cH9/l chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00210] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the HI 1 subtype (sometimes referred to herein as a "cHl 1/1 chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cHl 1/1 chimeric influenza hemagglutinin polypeptide is a cHl 1/1 chimeric influenza hemagglutinin polypeptide described in Table 8, above, or Section 6, below.

[00211] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H12 subtype (sometimes referred to herein as a "cH12/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH12/l chimeric influenza hemagglutinin polypeptide is a cH12/l chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00212] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H13 subtype (sometimes referred to herein as a "cH13/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH13/l chimeric influenza hemagglutinin polypeptide is a cH13/l chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00213] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza virus of the HI subtype and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H16 subtype (sometimes referred to herein as a "cH16/l chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH16/l chimeric influenza hemagglutinin polypeptide is a cH16/l chimeric influenza hemagglutinin polypeptide described in Table 8, above.

[00214] In a specific embodiment, a chimeric influenza virus HA polypeptide is a chimeric influenza virus HA polypeptide described in Table 8, above. In a specific embodiment, a chimeric influenza virus HA polypeptide is a chimeric influenza virus HA polypeptide described in Section 6, below.

5.1.1.2 A B Chimeric HA Polypeptides

[00215] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza B virus and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H5 subtype (sometimes referred to herein as a "cH5/B chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH5/B chimeric influenza hemagglutinin polypeptide is a cH5/B chimeric influenza hemagglutinin polypeptide described in Table 9 below.

[00216] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza B virus and (ii) the globular head domain of the hemagglutinin from an influenza virus of the H7 subtype (sometimes referred to herein as a "cH7/B chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cH7/B chimeric influenza hemagglutinin polypeptide is a cH7/B chimeric influenza hemagglutinin polypeptide described in Table 9 below.

[00217] Table 9. Exemplary cH5/B and cH7/B chimeric influenza hemagglutinin

polypeptides.

B B Chimeric HA Polypeptides

[00218] In certain embodiments, a chimeric influenza hemagglutinin (HA) polypeptide provided herein comprises (i) the stem domain of the hemagglutinin from an influenza B virus and (ii) the globular head domain of the hemagglutinin from a different influenza B virus strain (sometimes referred to herein as a "cB/B chimeric influenza hemagglutinin polypeptide"). In a specific embodiment, the cHB/B chimeric influenza hemagglutinin polypeptide is a cHB/B chimeric influenza hemagglutinin polypeptide described in Table 10 below.

[00219] Table 10. Exemplary cHB/B chimeric influenza hemagglutinin polypeptides. Chimeric influenza Strain of Influenza Virus HA Strain of Influenza Virus hemagglutinin Globular Head Domain HA Stem Domain polypeptide

cHB/B Any B other than B/Malaysia/2506/2004

B/Malaysia/2506/2004

cHB/B B/Lee/1940 B/Malaysia/2506/2004 cHB/B B/seal/Netherlands/1/99 HA (or a B/Malaysia/2506/2004

B/seal/Netherlands/l/99-like influenza

virus)

cHB/B Any B other than B/Florida/4/2006 B/Florida/4/2006

cHB/B B/Lee/1940 B/Florida/4/2006

cHB/B B/seal/Netherlands/1/99 HA (or a B/Florida/4/2006

B/seal/Netherlands/l/99-like influenza

virus)

cHB/B Any B other than B/Wisconsin/1/2010 B/Wisconsin/1/2010 cHB/B B/Lee/1940 B/Wisconsin/1/2010 cHB/B B/seal/Netherlands/1/99 HA (or a B/Wisconsin/1/2010

B/seal/Netherlands/l/99-like influenza

virus)

cHB/B Any B other than B/Brisbane/60/2008 B/Brisbane/60/2008 cHB/B B/Lee/1940 B/Brisbane/60/2008 cHB/B B/seal/Netherlands/1/99 HA (or a B/Brisbane/60/2008

B/seal/Netherlands/l/99-like influenza

virus)

5.1.2 Other Chimeric Influenza Virus HA Polypeptides

[00220] In another aspect, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid substitutions within an antigenic loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid residues in the loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In another aspect, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid substitutions within the 120 loop, 150 loop, 160 loop or 190 helix of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or more amino acid residues in the 120 loop, 150 loop, 160 loop or 190 helix of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In one embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In another embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In another embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising 2, 3, 4, 5 or more amino acid substitutions within 160 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In another embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In some embodiments, the influenza B virus is from the Yamagata lineage. In other embodiments, the influenza B virus is from the Victoria lineage. In specific embodiments, the influenza A virus from which the amino acid residues are derived for the amino acid

substitutions in one, two, three or more of the loops is an H5 (e.g., A/Vietnam/1203/04(HALo)), H8 (e.g., A/mallard/Sweden/24/2002), Hl l (e.g., A/northern shovel er/Netherlands/18/99), H12 strain (e.g., A_mallard_interior Alaska_7MP0167_2007), or HI 3 strain (e.g., A/black headed gull/Sweden/1/99). In specific embodiments, the amino acid residues TIP and

NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 11. In specific

embodiments, the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 12. In specific embodiments, the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 13. In specific embodiments, the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 14. In another specific embodiment, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and a 190 helix with the amino acid sequences of a 120 loop, a 150 loop, a 160 loop, and a 190 helix, respectively, set forth in Table 11, 12, 13 and 14. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 15. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix,

respectively, set forth in Table 16. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 17. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 18. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 19. In another specific embodiment, chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and 190 helix, respectively, set forth in Table 15, 16, 17, 18 or 19. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide, transmembrane domain, and cytoplasmic tail domain from the influenza B virus HA. In other embodiments, the chimeric HA polypeptide comprises the signal peptide from the influenza B virus HA but lacks the transmembrane and cytoplasmic tail domains. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide, transmembrane domain, and cytoplasmic tail domain from an influenza A virus HA. In other embodiments, the chimeric HA polypeptide comprises the signal peptide from an influenza A virus HA but lacks the transmembrane and cytoplasmic tail domains. In some embodiments, the chimeric HA comprises the signal peptide of the HA of the influenza virus backbone of the chimeric HA. For example, if the chimeric HA is engineered for an influenza A virus backbone (e.g., the influenza virus comprising or engineered to express the chimeric HA is an influenza A virus), then the chimeric HA comprises the signal peptide of the influenza A virus. In some embodiments, the chimeric HA comprises the signal peptide, transmembrane domain, and cytoplasmic domain of the HA of the influenza virus backbone of the chimeric HA. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide from the HA of the influenza virus that is engineered to express the chimeric HA. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide, transmembrane domain, and cytoplasmic tail domain from the HA of the influenza virus that is engineered to express the chimeric HA. Also provided herein are nucleic acids comprising nucleotide sequences encoding such a chimeric HA. In specific embodiments, the chimeric HA polypeptide is soluble. In certain embodiments, the chimeric HA polypeptides comprise 1, 2, 3, 4, 5 or more amino acid substitutions in the globular head domain of the influenza B virus HA which are outside of the 120 loop, 150 loop, 160 loop and/or 190 helix. For example, 1, 2, 3, or all of the following amino acids may be substituted with another amino acid residue: the last amino acid of the ectodomain of an influenza B virus HA may, amino acid 147 of influenza B virus HA (including the signal peptide), amino acid position 156 (glutamic acid) of the immature influenza

B/Yamagata/16/88 (substituted with, for example, lysine), and/or amino acid position 250 (glycine) of the immature influenza B/Yamagata/16/88 virus HA (substituted with, for example, glutamic acid).

[00221] Table 1 1. Exemplary substituted 120 loops sequences.

[00222] Table 12. Exemplary substituted 150 loops sequences.

PDKGASS (SEQ ID NO: 133) A/black headed gull/Sweden/ 1/99 (HI 3)

[00223] Table 13. Exemplary substituted 160 loops sequences.

KRGNQY (SEQ ID NO: 134) A/black headed gull/Sweden/ 1/99 (HI 3)

[00224] Table 14. Exemplary substituted 190 helix sequences.

190 helix original sequence in influenza B/Yamagata/16/88 virus:

NKNQMKN (SEQ ID NO: 112)

Substituted Sequence Strain (Subtype) used as a basis for the substituted sequence

NDAAMQT (SEQ ID NO: 113) A/Vietnam/1203/04 (HALo) (H5)

ADAKMQT (SEQ ID NO: 128) A Mallard/Sweden/24/2002 (H8)

TTLKMHQ (SEQ ID NO: 132) A/northern shovel er/Netherlands/18/99 (Hl l)

PTSDMQI (SEQ ID NO: 139) A/mallard/interior Alaska/7MP0167/2007 (H12)

VST MAK (SEQ ID NO: 135) A/black headed gull/Sweden/ 1/99 (HI 3)

[00225] Table 15. Exemplary 120 loop, 150 loop, 160 loop, and 190 helix substitutions based on amino acid residues from influenza A/Vietnam/1203/04 (HALo) (H5).

[00226] Table 16. Exemplary 120 loop, 150 loop, 160 loop, and 190 helix substitutions based on amino acid residues from influenza A/Mallard/Sweden/24/2002 (H8).

[00227] Table 17. Exemplary 120 loop, 150 loop, 160 loop, and 190 helix substitutions based on amino acid residues from influenza A/northern shovel er/Netherlands/18/99 (HI 1).

Loop/Helix Original Sequence in influenza Substituted Sequence with

B/Yamagata/16/88 virus amino acid residues from

A/northern

shoveler/Netherlands/18/99

120 loop TIP and LIP and

NIRLSTHNVINAERAPGGPYRL KIEL ST SNVINAEV APGGP YRL

(SEQ ID NO: 108) (SEQ ID NO: 129)

150 loop PNVTSRNG (SEQ ID NO: 123) PFGSSNS (SEQ ID NO: 130)

160 loop RDNKTA (SEQ ID NO: 110) HQSGTY (SEQ ID NO: 131)

190 helix NKNQMKN (SEQ ID NO: 112) TTLKMHQ (SEQ ID NO: 132) [00228] Table 18. Exemplary 120 loop, 150 loop, 160 loop, and 190 helix substitutions based on amino acid residues from influenza A/mallard/interior Alaska/7MP0167/2007 (H12).

[00229] Table 19. Exemplary 120 loop, 150 loop, 160 loop, and 190 helix substitutions based on amino acid residues from influenza A/black headed gull/Sweden/1/99 (H13).

[00230] In another aspect, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising one, two, three or all of the following: (i) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (ii) 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (iii) 2, 3, 4, 5 or more amino acid substitutions within the 160 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; and (iv) 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In some embodiments, the influenza B virus is from the Yamagata lineage. In other embodiments, the influenza B virus is from the Victoria lineage. In specific embodiments, the influenza A virus from which the amino acid residues are derived for the amino acid

NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 1 1. In specific

embodiments, the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 12. In specific embodiments, the amino acid residues RDNKTA (SEQ ID NO: 1 10) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 13. In specific embodiments, the amino acid residues NKNQMKN (SEQ ID NO: 1 12) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 14. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 15. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 16. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 17. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 18. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 19. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide, transmembrane domain, and cytoplasmic tail domain from the influenza B virus HA. In other embodiments, the chimeric HA polypeptide comprises the signal peptide from the influenza B virus HA but lacks the transmembrane and cytoplasmic tail domains. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide, transmembrane domain, and cytoplasmic tail domain from an influenza A virus HA. In other embodiments, the chimeric HA polypeptide comprises the signal peptide from an influenza A virus HA but lacks the transmembrane and cytoplasmic tail domains. In some embodiments, the chimeric HA comprises the signal peptide of the HA of the influenza virus backbone of the chimeric HA. For example, if the chimeric HA is engineered for an influenza A virus backbone (e.g., the influenza virus comprising or engineered to express the chimeric HA is an influenza A virus), then the chimeric HA comprises the signal peptide of the influenza A virus. In some embodiments, the chimeric HA comprises the signal peptide, transmembrane domain, and cytoplasmic domain of the HA of the influenza virus backbone of the chimeric HA. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide from the HA of the influenza virus that is engineered to express the chimeric HA. In some embodiments, the chimeric HA polypeptide may also comprise the signal peptide, transmembrane domain, and cytoplasmic tail domain from the HA of the influenza virus that is engineered to express the chimeric HA. In specific embodiments, the chimeric HA polypeptide is soluble. In certain embodiments, the chimeric HA polypeptides comprise 1, 2, 3, 4, 5 or more amino acid substitutions in the globular head domain of the influenza B virus HA which are outside of the 120 loop, 150 loop, 160 loop and/or 190 helix. For example, 1, 2, 3, or all of the following amino acids may be substituted with another amino acid residue: the last amino acid of the ectodomain of an influenza B virus HA may, amino acid 147 of influenza B virus HA (including the signal peptide), amino acid position 156 (glutamic acid) of the immature influenza B/Yamagata/16/88 (substituted with, for example, lysine), and/or amino acid position 250 (glycine) of the immature influenza B/Yamagata/16/88 virus HA (substituted with, for example, glutamic acid). In certain embodiments, the chimeric influenza virus HA polypeptide is a chimeric influenza virus HA polypeptide described in International Patent Application No.

PCT/US2017/037384 (International Patent Application Publication No. WO 2017/218624), which is incorporated by reference herein in its entirety.

[00231] In another aspect, provided herein are chimeric hemagglutinin (HA) polypeptides comprising (i) a hemagglutinin ectodomain from a first influenza B virus strain with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid substitutions within the 120 loop, 150 loop, 160 loop or 190 helix of the globular head domain of the influenza B virus HA and (ii) a signal peptide, a transmembrane domain and a cytoplasmic tail domain from a second influenza B virus strain, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid residues in the 120 loop, 150 loop, 160 loop or 190 helix of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In one embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising (i) a hemagglutinin ectodomain from an first influenza B virus strain with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the first influenza B virus strain and (ii) a signal peptide, a transmembrane domain and a cytoplasmic tail domain from a second influenza B virus strain, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In another embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising (i) a hemagglutinin ectodomain from an first influenza B virus strain with 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the first influenza B virus strain HA and (ii) a signal peptide, a transmembrane domain and a cytoplasmic tail domain from a second influenza B virus strain, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the in first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of a second influenza B virus strain HA. In another embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising (i) a hemagglutinin ectodomain from an first influenza B virus strain with 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the first influenza B virus strain HA and (ii) a signal peptide, a transmembrane domain and a cytoplasmic tail domain from a second influenza B virus strain, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the in first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus strain HA. In another embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising (i) a hemagglutinin ectodomain from a first influenza B virus strain with 2, 3, 4, 5 or more amino acid substitutions within the 160 loop of the globular head domain of the first influenza B virus strain HA and (ii) a signal peptide, a transmembrane domain and a cytoplasmic tail domain from a second influenza B virus strain, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In another embodiment, provided herein are chimeric hemagglutinin (HA) polypeptides comprising (i) a hemagglutinin ectodomain from a first influenza B virus strain with 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the globular head domain of the influenza B virus HA and (ii) a signal peptide, a transmembrane domain and a cytoplasmic tail domain from a second influenza B virus strain, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In some embodiments, the first influenza B virus strain and the second influenza B virus strain are from the same lineage. In some embodiments, the first influenza B virus strain and the second influenza B virus strain are from the same lineage but are different strains. In a specific embodiment, the first influenza B virus strain is the same strain as the second influenza B virus strain. In another embodiment, the first influenza B virus strain is a different strain than the second influenza B virus strain. In some embodiments, the first influenza B virus strain and the second influenza B virus strain are from different lineages. In some embodiments, the first influenza B virus strain is from the Yamagata lineage. In other embodiments, the first influenza B virus is from the Victoria lineage. In some embodiments, the second influenza B virus strain is from the Yamagata lineage. In other embodiments, the second influenza B virus is from the Victoria lineage. In a specific embodiment, the second influenza B virus strain is the same strain as the influenza virus backbone of an influenza virus either comprising, containing, or both the chimeric HA. In specific embodiments, the influenza A virus from which the amino acid residues are derived for the amino acid substitutions in one, two, three or more of the loops is an H5 (e.g., A/Vietnam/1203/04(HALo)), H8 (e.g.,

A/mallard/Sweden/24/2002), Hl l (e.g., A/northern shoveler/Netherlands/18/99), H12 strain (e.g., A_mallard_interior Alaska_7MP0167_2007), or HI 3 strain (e.g., A/black headed gull/Sweden/1/99). In specific embodiments, the amino acid residues TIP and

embodiments, the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 12. In specific embodiments, the amino acid residues RDNKTA (SEQ ID NO: 1 10) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 13. In specific embodiments, the amino acid residues NKNQMKN (SEQ ID NO: 1 12) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 14. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 15. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 16. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 17. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 18. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 19. In certain embodiments, the chimeric HA polypeptides comprise 1, 2, 3, 4, 5 or more amino acid substitutions in the globular head domain of the influenza B vims HA which are outside of the 120 loop, 150 loop, 160 loop and/or 190 helix. For example, 1, 2, 3, or all of the following amino acids may be substituted with another amino acid residue: the last amino acid of the ectodomain of an influenza B virus HA may, amino acid 147 of influenza B virus HA (including the signal peptide), amino acid position 156 (glutamic acid) of the immature influenza B/Yamagata/16/88 (substituted with, for example, lysine), and/or amino acid position 250 (glycine) of the immature influenza B/Yamagata/16/88 virus HA (substituted with, for example, glutamic acid).

[00232] In another aspect, provided herein are chimeric hemagglutinin (HA) polypeptides comprising: (a) a hemagglutinin ectodomain from a first influenza B virus strain with one, two, three or all of the following (i) 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the first influenza B virus strain HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (ii) 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the first influenza B virus strain HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (iii) 2, 3, 4, 5 or more amino acid substitutions within the 160 loop of the globular head domain of the first influenza B virus strain HA, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; and (iv) 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the globular head domain of the first influenza B virus strain HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the first influenza B virus strain HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; and (b) a signal peptide, a transmembrane domain and a cytoplasmic tail domain from a second influenza B virus strain. In some embodiments, the first influenza B virus strain and the second influenza B virus strain are from the same lineage. In some embodiments, the first influenza B virus strain and the second influenza B virus strain are from the same lineage but are different strains. In a specific embodiment, the first influenza B virus strain is the same strain as the second influenza B virus strain. In another embodiment, the first influenza B virus strain is a different strain than the second influenza B virus strain. In some embodiments, the first influenza B virus strain and the second influenza B virus strain are from different lineages. In some embodiments, the first influenza B virus strain is from the Yamagata lineage. In other embodiments, the first influenza B virus is from the Victoria lineage. In some embodiments, the second influenza B virus strain is from the Yamagata lineage. In other embodiments, the second influenza B virus is from the Victoria lineage. In a specific embodiment, the second influenza B virus strain is the same strain as the influenza virus backbone of an influenza virus either comprising, containing, or both the chimeric HA. In specific embodiments, the influenza A virus from which the amino acid residues are derived for the amino acid substitutions in one, two, three or more of the loops is an H5 (e.g., A/Vietnam/1203/04(HALo)), H8 (e.g.,

embodiments, the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 12. In specific embodiments, the amino acid residues RDNKTA (SEQ ID NO: 1 10) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 13. In specific embodiments, the amino acid residues NKNQMKN (SEQ ID NO: 1 12) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted an amino acid sequence of Table 14. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 15. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 16. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 17. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 18. In specific embodiments, a chimeric HA polypeptide described herein comprises a 120 loop, a 150 loop, a 160 loop, and/or a 190 helix with the amino acid sequences of the 120 loop, 150 loop, 160 loop, and/or 190 helix, respectively, set forth in Table 19. In certain embodiments, the chimeric HA polypeptides comprise 1, 2, 3, 4, 5 or more amino acid substitutions in the globular head domain of the influenza B virus HA which are outside of the 120 loop, 150 loop, 160 loop and/or 190 helix. For example, 1, 2, 3, or all of the following amino acids may be substituted with another amino acid residue: the last amino acid of the ectodomain of an influenza B virus HA may, amino acid 147 of influenza B virus HA (including the signal peptide), amino acid position 156 (glutamic acid) of the immature influenza B/Yamagata/16/88 (substituted with, for example, lysine), and/or amino acid position 250 (glycine) of the immature influenza B/Yamagata/16/88 virus HA (substituted with, for example, glutamic acid).

[00233] In another aspect, provided herein are chimeric hemagglutinin (HA) polypeptides comprising 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 polypeptide, wherein the HA stem domain polypeptide is from an influenza B virus HA, and wherein the HA globular head domain is the globular head domain derived from the influenza B virus HA and comprises one, two, three or all of the following: (a) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (b) 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; (c) 2, 3, 4, 5 or more amino acid substitutions within the 160 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; and (d) 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA. In specific embodiments, the chimeric influenza virus HA polypeptide further comprises the transmembrane domain and cytoplasmic tail domain of the influenza B virus HA. In specific embodiments, the influenza B virus is of the Yamagata lineage or of the Victoria lineage. In specific embodiments, the influenza B virus is influenza B/Yamagata/16/88. In specific embodiments, the influenza A virus is an influenza A virus of an H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18. In specific embodiments, the influenza A virus is an H5 HA subtype. In specific embodiments in which the influenza A virus is an H5 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRL STHNVINAERAPGGP YRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues FIP and

KIQLSTKNVINAEHAPGGPYRL (SEQ ID NO: 109); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PYQGKSS (SEQ ID NO: 124); (c) the acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KKNSTY (SEQ ID NO: 111); and/or (d) the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NDAAMQT (SEQ ID NO: 113) (i.e., the substituted amino acid sequences set forth in Table 15). In specific embodiments, the H5 subtype is influenza A/Vietnam/1203/04 (HALo) virus. In other specific embodiments, the influenza A virus is an H8 HA subtype. In specific embodiments in which the influenza A virus is an H8 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRL STHNVINAERAPGGP YRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues HIP and RIRLSTYNVINAETAPGGPYRL (SEQ ID NO: 125); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NASTGGQS (SEQ ID NO: 126); (c) the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KKKADTY (SEQ ID NO: 127); and/or (d) the amino acid residues

NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues ADAKMQT (SEQ ID NO: 128) (i.e., the substituted amino acid sequences set forth in Table 16). In specific embodiments, the influenza A virus H8 subtype is influenza A/Mallard/Sweden/24/2002 virus. In other specific embodiments, the influenza A virus is an HI 1 HA subtype. In specific embodiments in which the influenza A virus is an HI 1 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LIP and KIEL ST SNVINAEV APGGP YRL (SEQ ID NO: 129); (b) the amino acid residues

PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PFGSSNS (SEQ ID NO: 130); (c) the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues HQSGTY (SEQ ID NO: 131); and/or (d) the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of influenza B virus

B/Yamagata/16/88 are substituted with amino acid residues TTLKMHQ (SEQ ID NO: 132) (i.e., the substituted amino acid sequences set forth in Table 17). In specific embodiments, the HI 1 subtype is influenza A/northern shovel er/Netherlands/18/99 virus. In other specific

embodiments, the influenza A virus is an H12 HA subtype. In specific embodiments in which the influenza A virus is an H12 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and

NIRL STHNVINAERAPGGP YRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues YIP and

RIKLSTFNVINAETAPGGPYRL (SEQ ID NO: 136); (b) the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NNTSNQGS (SEQ ID NO: 137); (c) the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LKSGQF (SEQ ID NO: 138); and/or (d) the amino acid residues NKNQMKN (SEQ ID NO: 1 12) in the 190 helix of influenza B virus B/Yamagata/ 16/88 are substituted with amino acid residues PTSDMQI (SEQ ID NO: 139) (i.e., the substituted amino acid sequences set forth in Table 18). In specific embodiments, the H12 subtype is influenza A/mallard/interior Alaska/7MP0167/2007 virus. In other specific embodiments, the influenza A virus is an HI 3 HA subtype. In specific embodiments in which the influenza A virus is an HI 3 HA subtype, the HA globular head domain comprises one, two, three or all of the following: (a) the amino acid residues TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NIP and RIEL STHNVINAE VAPGGP YRL (SEQ ID NO: 168); (b) the amino acid residues

PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PDKGASS (SEQ ID NO: 133); (c) the amino acid residues RDNKTA (SEQ ID NO: 1 10) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KRGNQY (SEQ ID NO: 134); and/or (d) the amino acid residues NKNQMKN (SEQ ID NO: 1 12) in the 190 helix of influenza B virus

B/Yamagata/16/88 are substituted with amino acid residues VSTNMAK (SEQ ID NO: 135) (i.e., the substituted amino acid sequences set forth in Table 19). In specific embodiments, the H13 subtype is influenza A/black headed gull/Sweden/1/99 virus.

[00234] In another aspect, provided herein are chimeric hemagglutinin (HA) polypeptides comprising an HA ectodomain of an influenza B virus comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or more amino acid substitutions within an antigenic loop of the globular head domain of the influenza B virus HA (e.g., 120 loop, 150 loop, 160 loop and/or 190 helix), wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or more amino acid residues in the loop of the globular head of the influenza B virus HA with random amino acid residues that do not affect the conformation/structure of the HA.

[00235] The amino acid residues in the globular head domain of an influenza A virus HA in a region corresponding to an antigenic loop (e.g., 120 loop, 150 loop, 160 loop and/or 190 helix) in the globular head domain of an influenza B virus HA may be identified using techniques known to one skilled in the art. In specific embodiments, the amino acid residues in the globular head domain of an influenza A virus HA in a region corresponding to an antigenic loop (e.g., 120 loop, 150 loop, 160 loop and/or 190 helix) in the globular head domain of an influenza B virus HA are identified by comparing the amino acid sequences and/or structural information (e.g., crystal structures) of influenza A viruses and influenza B viruses. In particular embodiments, alignments of the amino acid sequences of HA of influenza A viruses and influenza B viruses as well as assessing the viruses for structural similarity enables the skilled person in the art to select the amino acid residues in the influenza B virus HA antigenic loop to substitute with amino acid residues from a corresponding region in the globular head domain of an influenza A virus HA. For example, one might want to refrain from substituting amino acid residues, such as cysteine, proline or both, in the influenza B virus HA antigenic loop that may impact the folding of the chimeric HA with amino acid residues from a corresponding region in the globular head domain of an influenza A virus HA. In addition, one might want to refrain from substituting amino acid residues in the influenza B virus HA antigenic loop that impact the coding for N-linked glycosylation sites (N-X-S/T). In selecting the amino acid residues to substitute, care should be taken to maintain the conformation/structure of the HA. In some embodiments, amino acid residues that are highly conserved in an antigenic loop of the globular head domain of influenza B virus HAs, one might want to refrain from substituting with amino acid residues from a corresponding region in the globular head domain of an influenza A virus HA. For example, those amino acid residues identified by Wang et al., 2008, Journal of Virology 82: 3011-3020 as being variant among influenza B viruses may be selected as amino acid residues within an antigenic loop of the globular head domain of an influenza B virus to substitute with other amino acid residues {e.g., other amino acid residues from a corresponding region of the globular head domain of an influenza A virus HA), while those amino acid residues within the antigenic loop of the globular head domain of an influenza B virus HA may not be substituted. In a specific embodiment, one, two or more amino acid residues identified by Wang et al, 2008, Journal of Virology 82: 3011-3020 in an antigenic loop of the globular head domain of HA as being variant among influenza B viruses may be selected as amino acid residues within an antigenic loop of the globular head domain of an influenza B virus HA to substitute with other amino acid residues {e.g., other amino acid residues from a corresponding region of the globular head domain of an influenza A virus HA). In specific embodiments, one may want to refrain from substituting any amino acid residues in an antigenic loop of the globular head domain of influenza B virus HAs identified as highly conserved. In a specific embodiment, when amino acid residues that are highly conserved in an antigenic loop of the globular head domain of influenza B virus HAs and amino acid residues in a corresponding region of the globular head domain of influenza A virus HAs, one might want to refrain from substituting with amino acid residues in the antigenic loop of the globular head domain of an influenza B virus HA with amino acid residues from a corresponding region in the globular head domain of an influenza A virus HA. For example, one of skill in the art may not want to substitute the methionine in the 190 helix of an influenza B virus with another amino acid residue. See, e.g., Section 6 below. In certain embodiments, with respect to amino acid residues such as proline found in an antigenic loop of the globular head domain of an influenza B virus HA, one might want to refrain from substituting with amino acid residues from a corresponding region in the globular head domain of an influenza A virus HA. In some embodiments, with respect to amino acid residues such as cysteine, proline or both found in an antigenic loop of the globular head domain of an influenza B virus HA, one might want to refrain from substituting with amino acid residues from a corresponding region in the globular head domain of an influenza A virus HA. In certain embodiments, one might want to refrain from substituting amino acid residues such as proline found in an antigenic loop of the globular head domain of an influenza B virus HA with amino acid residues from a corresponding region in the globular head domain of an influenza A virus HA. In specific embodiments, the amino acid residues substituted in an antigenic loop of the globular head domain of an influenza B virus are not consecutive amino acid residues. For example, amino acid residues that are found conformationally close to one another may be substituted for other amino acid residues. In other embodiments, the amino acid residues substituted in an antigenic loop of the globular head domain of an influenza B virus are consecutive amino acid residues. In certain embodiments, an amino acid residue found in the antigenic loop of an influenza B virus is substituted with a conservative amino acid residue {i.e., a conservative substitution). The effect of amino acid substitutions on the conformation/structure may be determined by assays known to one of skill in the art, e.g., structure programs, crystallography, or functional assays. See, e.g., Section 5.11 below, and Section 6 below. In a particular embodiment, the chimeric HA polypeptides may be evaluated for antigenic conservation using a panel of monoclonal antibodies that bind to conserved epitopes in the globular head domain of HA and the stem domain of HA. In a specific embodiment, the methods described in Section 6 below are used to evaluate antigenic conservation of the chimeric HA. In addition, the chimeric HA polypeptides described herein may be evaluated to determine whether the antigenic loops of the influenza B virus HA were mutated using techniques known to one of skill in the art or described herein (see, e.g., Section 6 below including the HI assay described therein). In particular, the chimeric HA polypeptides described herein may be evaluated to determine if the amino acid substitutions in the antigenic loop(s) of the influenza B virus HA result in loss of a variable region(s) of the influenza B virus HA using techniques known to one of skill in the art or described herein (see, e.g., Section 6 below including the HI assay described therein). In a specific embodiment, the chimeric HA polypeptides described herein may be evaluated to determine if the amino acid substitutions in the antigenic loop(s) of the influenza B virus HA reduce or eliminate the immunodominant epitopes of the influenza B virus HA using techniques known to one of skill in the art or described herein (see, e.g., Section 6 below, including the HI assay described therein). In a specific embodiment, a chimeric HA polypeptide described herein is assessed in an HI assay, such as described in Section 6 below to evaluate the replacement of the antigenic loop(s) in the influenza B virus HA.

[00236] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in site E of the globular head domain of an influenza A virus H3 HA. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20 or more amino acid residues in site Sa of the globular head domain of an influenza A virus HI HA. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20 or more amino acid residues in the 120 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in site Cb of the globular head domain of an influenza A virus HI HA. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in sites Sa and/or Cb of the globular head domain of an influenza A virus HI HA.

[00237] In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in site A of the globular head domain of an influenza A virus H3 HA. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in site Ca of the globular head domain of an influenza A virus HI HA.

[00238] In certain embodiments, 1, 2, 3, 4, 5 or more amino acid residues in the 160 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5 or more amino acid residues in site B of the globular head domain of an influenza A virus H3 HA. In some embodiments, 1, 2, 3, 4, 5 or more amino acid residues in the 160 loop of an influenza B virus HA are substituted with 1, 2, 3, 4, 5 or more amino acid residues in site Sa of the globular head domain of an influenza A virus HI HA.

[00239] In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in site B of the globular head domain of an influenza A virus H3 HA. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of an influenza B virus HA are substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in site Sb of the globular head domain of an influenza A virus HI HA.

[00240] In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18 subtype. In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18 subtype. In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the H5, H8, HI 1, H12, or H13 subtype. In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the H5 subtype (e.g,. A/Vietnam/1203/04). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the H8 subtype (e.g.,

A/mallard/Sweden/24/02). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the HI 1 subtype (e.g., A/Northern shoveler/Netherlands/18/99). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the H12 subtype. In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the H13 subtype (e.g., A/black headed gull/Sweden/1/99). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza virus of the H17 subtype (e.g., A/yellow shouldered bat/Guatamela). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an avian influenza virus. In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza

A/mallard/Sweden/24/2002 virus (GenBank Accession No. CY060249.1; GenBank GI No. 294441479; see, also, FIG. 20A and FIG. 20B). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza A/Vietnam/1203/04 virus (GenBank Accession No. EF541403.1; GenBank GI No. 145284465; see, also, FIG. 21 A and FIG. 21B and Steel et al, 2009, Journal of Virology, 83(4): 1742-1753 for the HA of influenza A/Vietnam/1203/04 (HALo) virus). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza A/northern shovel er/Netherlands/18/99 virus

(GenBank Accession No. CY060417.1; GenBank GI No. 294441876; see, also, FIG. 22A and FIG. 22B). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza A mallard interior Alaska_7MP0167_2007 virus (GenBank Accession No. CY077198.1; GenBank GI No.

312652817; see, also, FIG. 23A and FIG. 23B). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza A/Puerto Rico/8/34 virus (GenBank Accession No. AF389118.1; GenBank GI No. 21693168; see, also, FIG. 24A and FIG. 24B). In a specific embodiment, the influenza A virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza A/black headed gull/Sweden/1/99 (GenBank Accession No. AY684887.1; see, also, FIG. 40A and 40B). In a specific embodiment, the influenza B virus HA utilized in the generation of a chimeric HA polypeptide described herein is an HA from an influenza B virus of the Yamagata lineage. In a specific embodiment, the influenza B virus HA utilized in the generation of a chimeric HA polypeptide described herein is an HA from an influenza B virus of the Victoria lineage. In a specific embodiment, the influenza B vims HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza

B/Yamagata/16/88 virus (see, FIG. 25A and FIG. 25B). In a specific embodiment, the influenza B virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from an influenza B/Malaysia/2506/04 mouse adapted (MA) virus (see, e.g., SEQ ID NO: 144 and 151). In a specific embodiment, the influenza B virus HA utilized in the generation of a chimeric HA polypeptide described herein is the HA from influenza B/Malaysia/2506/04 virus (see, e.g., GenBank Accession No. CY040449.1).

[00241] In a specific embodiment, a chimeric HA polypeptide is a chimeric HA polypeptide described in Section 6 below. In a specific embodiment, a chimeric HA polypeptide comprises the amino acid sequence of the chimeric HA polypeptide in Figure 29, 31, 33, or 35. In another specific embodiment, a chimeric HA polypeptide comprises the amino acid sequence of the chimeric HA polypeptide in Figure 29, 31, 33, or 35 without the signal peptide. In another specific embodiment, a chimeric HA polypeptide comprises the amino acid sequences of the ectodomain of the chimeric HA polypeptide in Figure 29, 31, 33, or 35.

[00242] In a specific embodiment, the influenza B virus HA sequence utilized to generate a chimeric HA polypeptide described herein is the HA sequence from an influenza B virus described in Section 5.6 below. In a specific embodiment, the influenza B virus HA sequence utilized to generate a chimeric HA polypeptide described herein is the HA sequence from an influenza B virus described in Section 6 below. In a specific embodiment, the influenza A virus HA sequence utilized to generate a chimeric HA polypeptide described herein is the HA sequence from an influenza A virus described in Section 5.6 below. In a specific embodiment, the influenza A virus HA sequence utilized to generate a chimeric HA polypeptide described herein is the HA sequence from an influenza A virus described in Section 6 below. For example, the influenza A virus HA may be from a group 1 or a group 2 virus. In specific embodiments, the influenza A virus HA is from an HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, or HI 7 influenza A virus.

[00243] In a specific embodiment, a chimeric influenza virus HA polypeptide is one described in International Patent Application No. PCT/US2017/037384, filed June 14, 2017 (now

International Patent Application Publication No. WO 2017/218624), which is incorporated by reference herein in its entirety. 5.2 INFLUENZA HEMAGGLUTININ HEAD DOMAIN POLYPEPTIDES

[00244] Provided herein are influenza hemagglutinin head domain polypeptides for use in the generation of the chimeric influenza virus hemagglutinin polypeptides described herein in, e.g., Section 5.1 above (in particular, Section 5.1.1 above).

[00245] Generally, the influenza hemagglutinin head domain polypeptides provided herein are polypeptides that comprise or consist essentially of the globular head domain of an influenza hemagglutinin polypeptide. The head domain of an influenza hemagglutinin polypeptide is the head domain that is generally recognized by those of skill in the art.

[00246] In certain embodiments, the influenza hemagglutinin head domain polypeptides provided herein comprise an influenza hemagglutinin head domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, or 99% amino acid sequence identity to an influenza hemagglutinin head domain known to those of skill in the art.

[00247] Also provided herein are influenza hemagglutinin head domain polypeptides comprising amino acids from two or more strains or subtypes of influenza virus. In certain embodiments, a chimeric HAl subunit comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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. In certain embodiments, a chimeric 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 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. In certain embodiments, the amino acids from the first influenza virus strain or subtype can be consecutive, and/or can represent portions of the N- and/or C-termini of a chimeric HAl domain.

[00248] Also provided herein are 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, 115, 110, 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. Also provided herein are influenza hemagglutinin head domain polypeptides comprising deleted forms of a known influenza hemagglutinin head domain, wherein about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-1 10, 1 10-120, 120-130, 130-140, or 140-150 amino acid residues are deleted from the head domain.

[00249] In certain embodiments, the influenza HA globular head domain of a chimeric HA comprises one, two, three, or more heterologous antigenic regions. In one embodiment, the 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). In a specific embodiment, the 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. In accordance with this embodiment, the one, two, three or more NA antigenic regions may replace one, two, three or more HA antigenic regions. In another specific embodiment, the influenza HA globular head domain of a chimeric HA comprises the amino acid sequence ILRTQESEC (SEQ ID NO: 189), which is located between residues 222 and 230 (N2 numbering) in the enzymatic active site of NA. In certain embodiments, this amino acid sequence replaces one, two, three or more antigenic regions of the HA globular head domain of a chimeric HA. For example, 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. In another example, 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 .

[00250] Provided herein are 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. Also provided herein are 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.

[00251] Also provided herein are influenza hemagglutinin head domain polypeptides comprising a deletion 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 hemagglutinin head domain (e.g., antigenic sites A, B, C, and D, wherein the head domain is from subtype H3 or antigenic sites Sa, Sb, Ca and Cb, wherein the head domain is from subtype HI). In a specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a deletion of one antigenic region (e.g., a region of the head domain known to comprise or consist of an epitope). In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a deletion of two antigenic regions (e.g., two regions of the head domain known to comprise or consist of an epitope). In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a deletion of three antigenic regions (e.g., three regions of the head domain known to comprise or consist of an epitope). In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a deletion of four antigenic regions (e.g., four regions of the head domain known to comprise or consist of an epitope). In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a deletion of five antigenic regions (e.g., five regions of the head domain known to comprise or consist of an epitope). Those of skill in the art can readily determine the antigenic regions (e.g., epitopes) of influenza head domains known in the art or later identified using techniques known to those of skill in the art and described herein.

[00252] In certain embodiments, the influenza hemagglutinin head domain polypeptides of the chimeric influenza virus hemagglutinin polypeptides described herein comprise (i) one, two, three, or more antigenic regions from an influenza hemagglutinin head domain polypeptide that are homologous to the stem domain (i.e., derived from the same influenza virus strain or subtype) and (ii) one, two, three, or more antigenic regions from an influenza hemagglutinin head domain polypeptide that are heterologous to the stem domain (i.e., derived from a different influenza virus strain or subtype). In a specific embodiment, the C antigenic site/region of the head domain is homologous to the stem domain (i.e., derived from the same influenza virus strain or subtype). In another specific embodiment, the D antigenic site/region of the head domain is homologous to the stem domain (i.e., derived from the same influenza virus strain or subtype). In another specific embodiment, the C and D antigenic sites/regions of the head domain are homologous to the stem domain (i.e., derived from the same influenza virus strain or subtype). In yet another specific embodiment, the Ca and/or Cb antigenic sites/regions of the head domain are homologous to the stem domain (i.e., derived from the same influenza virus strain or subtype). [00253] Also provided herein are influenza hemagglutinin head domain polypeptides comprising a replacement 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 hemagglutinin head domain with a non-antigenic polypeptide sequence (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 a specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a replacement of one antigenic region (e.g., a region of the head domain known to comprise or consist of an epitope) with a non-antigenic polypeptide sequence (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 another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide

comprising a replacement of two antigenic regions (e.g., two regions of the head domain known to comprise or consist of an epitope) with non-antigenic polypeptide sequences (e.g., polypeptide sequences that are known to not induce an immune response or are known to generate an immune response that is not specific to influenza). In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a replacement of three antigenic regions (e.g., three regions of the head domain known to comprise or consist of an epitope) with non-antigenic polypeptide sequences (e.g., polypeptide sequences that are known to not induce an immune response or are known to generate an immune response that is not specific to influenza). In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a replacement of four antigenic regions (e.g., four regions of the head domain known to comprise or consist of an epitope) with non- antigenic polypeptide sequences (e.g., polypeptide sequences that are known to not induce an immune response or are known to generate an immune response that is not specific to influenza). In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising a replacement of five antigenic regions (e.g., five regions of the head domain known to comprise or consist of an epitope) with non-antigenic polypeptide sequences (e.g., polypeptide sequences that are known to not induce an immune response or are known to generate an immune response that is not specific to influenza). Those of skill in the art can readily determine the antigenic regions (e.g., epitopes) of influenza head domains known in the art or later identified using techniques known to those of skill in the art and described herein. [00254] In another specific embodiment, provided herein is an influenza hemagglutinin head domain polypeptide comprising one, two, three, or more heterologous antigenic regions, i.e., one, two, three, or more antigenic regions from the hemagglutinin 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).

[00255] The influenza hemagglutinin head domain polypeptides provided herein might be based on {i.e. might have sequence identity to) the head domain of any influenza hemagglutinin known to those of skill or later discovered. In certain embodiments, influenza hemagglutinin head domain polypeptides are based on the head domain of an influenza A hemagglutinin {e.g., the head domain of the hemagglutinin of an influenza A virus described in Section 5.6 below). In certain embodiments, the 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. In certain embodiments, 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 5.1 above or 5.6 below). In a specific embodiment, the influenza hemagglutinin head domain polypeptides are based on the HA head domain of

B/Yamagata/16/88. In some embodiments, the influenza hemagglutinin head domain

polypeptides are based on the HA head domain of B/Seal/Netherlands/1/99. In a specific embodiment, the influenza hemagglutinin head domain polypeptides are based on the head domain of an influenza A hemagglutinin selected from an H5, H6, and/or H9 subtype. In another specific embodiment, the influenza hemagglutinin head domain polypeptides are based on the head domain of an influenza A hemagglutinin selected from an H5, H7, and/or H9 subtype. In a specific embodiment, the influenza hemagglutinin head domain polypeptides are based on the head domain of an influenza A hemagglutinin selected from an H5, H8, and/or H9 subtype. In another specific embodiment, the influenza hemagglutinin head domain

polypeptides are based on the head domain of an influenza A hemagglutinin from an H5, H7, H8, H11, or H12 subtype. 5.3 INFLUENZA HEMAGGLUTININ STEM DOMAIN POLYPEPTIDES

[00256] Provided herein are influenza hemagglutinin stem domain polypeptides for use in the generation of chimeric influenza virus hemagglutinin polypeptides described herein in, e.g., Section 5.1 above (in particular, Section 5.1.1 above). While not intending to be bound by any particular theory of operation, it is believed that, in the context of the chimeric influenza virus hemagglutinin polypeptides provided herein, the influenza hemagglutinin stem domain polypeptides are useful for presenting one or more relatively conserved antigenic regions to a host immune system in order to generate an immune response that is capable of cross-reacting with a plurality of influenza strains. Since the one or more antigenic regions are well conserved across influenza hemagglutinin subtypes, such an immune response might cross-react with several subtypes of full-length influenza hemagglutinin polypeptides.

[00257] Generally, the influenza hemagglutinin stem domain polypeptides provided herein are polypeptides that comprise or consist essentially of the stem domain of an influenza

hemagglutinin polypeptide. The stem domain of an influenza hemagglutinin polypeptide is the stem domain that is generally recognized by those of skill in the art. In certain embodiments, the influenza hemagglutinin stem domain polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96% or 98%) amino acid sequence identity to an influenza hemagglutinin stem domain known to those of skill in the art. In certain embodiments, the influenza hemagglutinin stem domain polypeptide has at least 90% amino acid sequence identity to an influenza hemagglutinin stem domain known to those of skill in the art. In certain embodiments, the influenza hemagglutinin stem domain polypeptide has at least 95% amino acid sequence identity to an influenza hemagglutinin stem domain known to those of skill in the art. In certain embodiments, the influenza hemagglutinin stem domain polypeptide has at least 96% amino acid sequence identity to an influenza hemagglutinin stem domain known to those of skill in the art. In certain embodiments, the influenza hemagglutinin stem domain polypeptide has at least 98% amino acid sequence identity to an influenza hemagglutinin stem domain known to those of skill in the art.

[00258] In certain embodiments, the influenza hemagglutinin stem domain polypeptides provided herein comprise little or no globular head domain of an influenza hemagglutinin polypeptide. In certain embodiments, the influenza hemagglutinin stem domain polypeptides provided herein comprise only 1-15, 1-10, 1-5, 5-10, 5-15, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues of the globular head domain of an influenza hemagglutinin polypeptide. In certain embodiments, an influenza hemagglutinin stem domain polypeptide is an influenza hemagglutinin that has had its globular head domain deleted by any technique deemed suitable by one of skill in the art.

[00259] In certain embodiments, influenza hemagglutinin stem domain polypeptides described herein maintain the cysteine residues identified in influenza hemagglutinin

polypeptides as Ap and Aq in Fig. 1. In certain embodiments, influenza hemagglutinin stem domain polypeptides described herein maintain the alanine residues identified in influenza B virus hemagglutinin polypeptides as D_p and D_q. In certain embodiments, influenza

hemagglutinin stem domain polypeptides described herein substitute the each of the alanine residues identified in influenza B virus hemagglutinin polypeptides as D_p and D_q with cysteine residues. In certain embodiments, influenza hemagglutinin stem domain polypeptides described herein have greater stability at a pH lower than the hemagglutinin of a wild-type influenza virus (e.g., a pH less than 5.2, less than 5.1, less than 5.0, or less than 4.9, such as 4.8, 4.7, 4.6, 4.5, 4.4., 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, etc.). In particular embodiments, influenza hemagglutinin stem domain polypeptides described herein undergo conformational changes from the pre-fusion to the fusion conformation at a pH lower than the hemagglutinin of wild-type influenza viruses. In some embodiments, influenza hemagglutinin stem domain polypeptides described herein comprise one or more amino acid substitutions, such as HAl H17Y (H3 numbering) that increases the stability of the polypeptides at a low pH (e.g., a pH of between 4.9 to 5.2, 4.5 to 3.5, 3.5 to 2.5, 2.5 to 1.5, 1.5 to 0.5). The stability of 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.

[00260] The influenza hemagglutinin stem domain polypeptides can be prepared according to any technique deemed suitable to one of skill in the art, including techniques described below. In certain embodiments, the stem domain polypeptides are isolated.

[00261] In specific embodiments, the primary structure of an influenza hemagglutinin stem domain polypeptide comprises, in the following order: an HAl N-terminal stem segment, an HAl C-terminal stem segment and an HA2 stem domain. In some embodiments, the entire HA2 domain is present. [00262] The primary sequence might be formed by a single polypeptide, or it might 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. As is known to those of skill in the art, a single HA polypeptide might be cleaved, for example by a protease, under appropriate expression conditions to yield two polypeptides in quaternary association. The cleavage is typically between the HAl C- terminal stem segment and the HA2. In certain embodiments, provided herein are multiple polypeptide, for example two polypeptide, influenza hemagglutinin stem domains. In multiple polypeptide embodiments, the HAl segments and HA2 are in quaternary association.

[00263] In certain embodiments, 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.

[00264] Influenza hemagglutinin HA2 typically comprises a stem domain, transmembrane domain and a cytoplasmic domain. In certain embodiments, provided herein are influenza hemagglutinin stem domain polypeptides that comprise an HA2 stem domain, an HA2 luminal domain, an HA2 transmembrane domain and an HA2 cytoplasmic domain. Such influenza hemagglutinin stem domain polypeptides might be expressed as membrane-bound antigens. In certain embodiments, provided herein are influenza hemagglutinin 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. Such influenza hemagglutinin stem domain polypeptides might be expressed as membrane-bound antigens. In certain embodiments, provided herein are influenza hemagglutinin 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. Such influenza hemagglutinin stem domain polypeptides might advantageously be expressed as soluble polypeptides. In certain embodiments, provided herein are influenza hemagglutinin stem domain polypeptides that comprise an HA2 stem domain but lack an HA2 luminal domain, an HA2 transmembrane domain and an HA2 cytoplasmic domain. Such influenza hemagglutinin stem domain polypeptides might advantageously be expressed as soluble polypeptides. In certain

embodiments, the 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, and 2013/0129761, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 which are incorporated herein by reference in their entireties.

[00265] Also provided herein 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. Further provided herein are 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.

[00266] In some embodiments, 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 a stem domain portion of 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. Typically, an influenza A virus 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 according to H3 numbering. This cysteine residue, termed A_p herein, is generally capable of forming a disulfide bridge with a cysteine residue in the C-terminal stem segment of HAl . Sequences of 16 representative influenza A hemagglutinins are presented in Fig. 1, and residue A_p is identified in each. Typically, an influenza B virus 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 57^th residue of the HAl according to B/Yamagata/16/88 numbering.

[00267] In certain embodiments, the influenza B virus hemagglutinin stem domain

polypeptides of the chimeric influenza virus hemagglutinin polypeptides described herein donot maintain the alanine residues identified in influenza hemagglutinin polypeptides as D_p and D_q; rather, D_p and D_q are each substituted with a cysteine residue. Thus, in certain embodiments, in the primary sequence of a chimeric influenza virus hemagglutinin polypeptide described herein: (i) the N-terminal segment of an influenza B virus hemagglutinin stem domain polypeptide ends at the location of the alanine residue corresponding to D_p, wherein the alanine residue corresponding to Dp is substituted with a cysteine residue, (ii) the C-terminal segment of an influenza B virus hemagglutinin stem domain polypeptide begins at the location of the alanine residue corresponding to D_q, wherein the alanines residue corresponding to D_q is substituted with a cysteine residue; and (iii) the influenza A virus hemagglutinin head domain polypeptide is between the HAl N-terminal and HAl C-terminal stem segments of the influenza B virus hemagglutinin stem domain polypeptide.

[00268] In certain embodiments, the HAl N-terminal stem segment does not end exactly at A_p (e.g. , Cys52 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_p. For example, in certain embodiments, the HAl N-terminal stem segment ends at A_p-i, A_p-₂, A_p-₃, A_p-4, A_p-5, A_p-6, A_p-7, Ap-8, A_p-9, A_p-io, A_p-

11, A_p-12, A_p-13, A_p-14, A_p-15, A_p-16, A_p-17, A_p-18, A_p-19, A_p-20, A_p-21, 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-3o. In certain embodiments, the HAl N-terminal stem segment of the chimeric hemagglutinin polypeptides described herein ends in the range of A_p-i to A_p-3, A_p-3 to A_p-5, A_p-5 to A_p-8, A_p-8 to A_p-io, A_p-io to A_p-i5, A_p-i5 to A_p-2o, A_p-2o to A_p-3o, A_p-3o to A_p-4o. In other embodiments, the HAl N-terminal stem segment ends at A_p+i, A_p+2, A_p+3, A_p+4, A_p+5, A_p+6, A_p+7,

A_p+8, A_p+9, A_p+10, A_p+ll, A_p+12, A_p+13, A_p+14, A_p+15, A_p+16, A_p+17, A_p+18, A_p+19, A_p+20, A_p+21, A_p+22, A_p+23, A_p+24, A_p+25, A_p+26, A_p+27, A_p+28, A_p+29, A_p+30, A_p+31, A_p+32, A_p+33, A_p+34, A_p+35, A_p+36, A_p+37,

A_p+38, A_p+39, A_p+4o. In certain embodiments, the HAl N-terminal stem segment of the chimeric hemagglutinin polypeptides described herein ends in the range of A_p+i to A_p+5, A_p+5 to A_p+io,

A_p+10 tO A_p+15, A_p+15 tO A_p+20, A_p+20 tO A_p+25, A_p+25 tO A_p+30, A_p+30 tO A_p+35, A_p+35 tO A_p+40, Or A_p+40 to Ap+50. 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 globular head domain so that the resulting chimeric influenza virus HA polypeptide is capable of forming a three-dimensional structure similar to an HA polypeptide of a wild-type influenza virus. In such embodiments, an influenza hemagglutinin head domain polypeptide (which is heterologous to the influenza hemagglutinin stem domain polypeptide) is located, in primary sequence, between the N-terminal and C- terminal segments of the influenza hemagglutinin stem domain polypeptide.

[00269] In certain embodiments, the HAl N-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein does not end exactly at D_p (e.g., Alas7 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering), but at a residue in sequence and structural vicinity to D_p. For example, in certain embodiments, the HAl N-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein ends at D_p-i, D_p-2, D_p-3, D_p-4, D_p-5, D_p-6, D_p-7, D_p-₈, D_p-9, D_p-io, D_p-n, Dp-12, D_p-i₃,

Dp-14, Dp-15, Dp-16, Dp-17, Dp-1₈, Dp-19, Dp-20, Dp-21, Dp-22, Dp-23, Dp-23, Dp-24, Dp-25, Dp-26, Dp-27, Dp-2₈,

Dp-29, Dp-3o. In certain embodiments, the HAl N-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein ends in the range of D_p-i to D_p-3, D_p- 3 to Dp-5, Dp-5 to D_p-₈, D_p-₈ to Dp-io, Dp-io to D_p-is, D_p-is to Dp-20, Dp-20 to Dp-30, Dp-30 to D_p-4o. For example, an HAl N-terminal stem segment ending at D_p-io would end at the amino acid position Leu47 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering. In certain embodiments, the HAl N-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein ends at D_p+i, D_p+2, D_p+3, D_p+4, D_p+5,

Dp+6, Dp+7, Dp+₈, Dp+9, Dp+10, Dp+11, Dp+12, Dp+13, Dp+14, Dp+15, Dp+16, Dp+17, Dp+1₈, Dp+19, Dp+20, Dp+21, Dp+22, Dp+23, Dp+24, Dp+25, Dp+26, Dp+27, Dp+2₈, Dp+29, Dp+30, Dp+31, Dp+32, Dp+33, Dp+34, Dp+35,

Dp+36, Dp+37, D_p+3₈, Dp+39, Dp+40. In certain embodiments, the HAl N-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein ends in the range of

Dp+l tO Dp+5, Dp+5 tO Dp+10, Dp+10 tO Dp+15, Dp+15 tO Dp+20, Dp+20 tO Dp+25, Dp+25 tO Dp+30, Dp+30 to

Dp+35, Dp+35 to Dp+40, or Dp+40 to Dp+50. For example, an HAl N-terminal stem segment ending at Dp+25 would end at Leu82 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering. 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 A

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. In such embodiments, an influenza A virus hemagglutinin head domain polypeptide is located, in primary sequence, between the N-terminal and C-terminal segments of the influenza hemagglutinin stem domain polypeptide.

[00270] In certain embodiments, the influenza hemagglutinin stem domain polypeptides comprise an HAl N-terminal stem segment having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%) or 98%) amino acid sequence identity to an influenza HAl N-terminal stem segment known to those of skill in the art. Exemplary known HAl N-terminal stem segments are provided in the tables disclosed in International Publication No. WO 2010/1 17786, WO 201 1/123495, and WO 2013/043729, U.S. Publication Nos. 2010/0297174, and 2013/0129761, and U.S. Application No. 14/345,816, which published as U. S. Patent Publication No. 2015/0132330 which are incorporated herein by reference in their entireties.

[00271] Also provided herein are influenza hemagglutinin stem domain polypeptides comprising deleted forms of HAl N-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 N-terminal stem segment. Also provided herein are influenza hemagglutinin stem domain polypeptides comprising deleted forms of a known influenza hemagglutinin stem domain, wherein about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100 amino acid residues are deleted from the stem domain. In certain embodiments, provided herein are influenza hemagglutinin stem domain polypeptides that comprise expanded forms of HAl N-terminal 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 stem segments; these added residues might be derived from the amino acid sequence of a globular head domain adjacent to an HAl N-terminal stem segment. Further provided herein are influenza

hemagglutinin stem domain polypeptides comprising altered forms of HAl N-terminal stem segments wherein up to 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. Also provided herein are influenza hemagglutinin stem domain polypeptides comprising altered forms of a known influenza hemagglutinin stem 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 stem domain are substituted (e.g., conservatively substituted) with other amino acids. Further provided are influenza hemagglutinin stem domain polypeptides comprising deleted and altered HAl N-terminal stem segments. In certain embodiments, up to 50, 60, or more amino acids are deleted from the N- terminus of an influenza hemagglutinin stem domain (as viewed from the primary amino acid sequence) and up to 70, 80, or more amino acids are deleted from the C-terminus of an influenza hemagglutinin stem domain (as viewed from the primary amino acid sequence).

[00272] 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. Typically, an influenza A virus 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. 1, and residue A_q is identified in each. Typically, an influenza B virus HAl C- terminal stem segment corresponds to a polypeptide consisting of the alanine residue located in sequence at approximately the 306^th residue of an HAl (using /Yamagata/16/88 numbering) through the C-terminal amino acid of the HAl .

[00273] In certain embodiments, the HAl C-terminal stem segment does not start at A_q (e.g., Cys277 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. For example, in certain embodiments, 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, A_q-

11, A_q-12, A_q-13, A_q-14, A_q-15, A_q-20, A_q-25, A_q-30, A_q-35, A_q-40, A_q-45, A_q-50, A_q-55, A_q-60, A_q-65, A_q-70,

A_q-75, or A_q-8o. In certain embodiments, the HAl C-terminal stem segment starts at in the range of A_q-i to A_q-5, A_q-5 to A_q-io, A_q-io to A_q-i5, A_q-i5 to A_q-2o, A_q-2o to A_q-25, A_q-25 to A_q-3o, A_q-3o 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-7o, A_q-75 to A_q-8o. In other embodiments, the HAl C-terminal stem segment starts at 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, A_q+is, A_q+₂o, A_q+₂₅, or A_q+₃o. In certain embodiments, the HAl C-terminal stem segment of the chimeric hemagglutinin polypeptides described herein starts in the range of A_q+i to A_q+₃, A_q+₃ to A_q+5, A_q+5 to A_q+₈, A_q+₈ to A_q+io, A_q+io to A_q+i5, or A_q+i5 to A_q+₂o. 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 globular head domain so that the resulting chimeric influenza virus HA polypeptide is capable of forming a three- dimensional structure similar to a wild-type influenza virus HA. In certain embodiments, the influenza hemagglutinin stem domain polypeptides of the chimeric influenza virus

hemagglutinin polypeptides described herein maintain the cysteine residues identified in influenza hemagglutinin polypeptides as Ap and Aq in Fig. 1, i.e., the cysteine residues identified in influenza hemagglutinin polypeptides as Ap and Aq in Fig. 1 are maintained in the chimeric influenza virus hemagglutinin polypeptides described herein.

[00274] In certain embodiments, the HAl C-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein does not start at D_q {i.e., Ala₃06 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering), but at a residue in sequence and structural vicinity to D_q. For example, in certain embodiments, the HAl C-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein starts at about D_q-i, D_q-2, D_q-3, D_q-4, D_q-5, D_q-6, D_q-7, D_q-8, D_q-9, D_q-io, D_q-n, D_q-i₂, D_q-i₃, D_q-i4, D_q-i5, D_q-2o, D_q-25, D_q-3o, D_q-35, D_q-4o, D_q-45, D_q-5o, D_q-55, D_q-6o, D_q-65, D_q-7o, D_q-75, or D_q-8o. In certain embodiments, the HAl C-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein starts in the range of D_q-i to D_q-5, D_q-5 to D_q-io, D_q-io tO D_q-15, D_q-15 tO D_q-20, D_q-20 tO D_q-25, D_q-25 tO D_q-30, D_q-30 tO D_q-35, D_q-35 toD_q-40, D_q-40 tO D_q-45, D_q-

45 to D_q-5o, D_q-5o to D_q-55, D_q-55 to D_q-6o, D_q-6o to D_q-65, D_q-65 toD_q-7o, D_q-75 to D_q-8o. For example, an HAl C-terminal stem segment ending at D_q-2 would start at Gly304 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering; and an HAl C-terminal stem segment ending at D_q-io would start at Isoleucine296 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering. In certain embodiments, the HAl C- terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein starts at D_q+i, D_q+₂, D_q+₃, D_q+4, D_q+5, D_q+6, D_q+7, D_q+₈, D_q+9, D_q+io, D_q+n, D_q+i₂, D_q+i₃,

D_q+14, D_q+15, D_q+16, D_q+17, D_q+1₈, D_q+19, D_q+20, D_q+21, D_q+22, D_q+23, D_q+24, D_q+25, D_q+26, D_q+27, D_q+₂₈,

D_q+29, D_q+₃o. In certain embodiments, the HAl C-terminal stem segment of the chimeric influenza virus hemagglutinin polypeptides described herein starts in the range of D_q+i to D_q+₃, D_q+₃ toD_q+5, D_q+5 to D_q+₈, D_q+₈ to D_q+io, D_q+io to D_q+is, or D_q+is to D_q+₂o. 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 influenza A virus 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. In such embodiments, an influenza A virus hemagglutinin head domain polypeptide is located, in primary sequence, between the N-terminal and C-terminal segments of the influenza hemagglutinin stem domain polypeptide.

[00275] In certain embodiments, the influenza hemagglutinin stem domain polypeptides comprise an HAl C-terminal stem segment having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%) or 98%) amino acid sequence identity to an influenza HAl C-terminal stem segment known to those of skill in the art. Exemplary known HAl C-terminal 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, and 2013/0129761, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 which are incorporated herein by reference in their entireties.

[00276] In certain embodiments, 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. In certain embodiments, the end of the N-terminal stem segment is A_p-₂, and the start of the C-terminal stem segment is Aq-₂. In certain embodiments, the end of the N-terminal stem segment is A_p-₃, and the start of the C-terminal stem segment is Aq-₃. In certain embodiments, the end of the N-terminal stem segment is A_p-4, and the start of the C- terminal stem segment is Aq-₄. In certain embodiments, the end of the N-terminal stem segment is Ap-5, and the start of the C-terminal stem segment is A_q-5.

[00277] In certain embodiments, 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. In certain embodiments, the end of the N-terminal stem segment is A_p+2, and the start of the C-terminal stem segment is A_q+2. In certain embodiments, the end of the N-terminal stem segment is A_p+₃, and the start of the C-terminal stem segment is A_q+3. In certain embodiments, the end of the N-terminal stem segment is A_p+₄, and the start of the C-terminal stem segment is A_q+₄. In certain embodiments, the end of the N-terminal stem segment is A_p+₅, and the start of the C-terminal stem segment is A_q+₅.

[00278] In certain embodiments, 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. In certain embodiments, the end of the N-terminal stem segment is A_p-₂, and the start of the C-terminal stem segment is A_q+2. In certain embodiments, the end of the N-terminal stem segment is A_p-₃, and the start of the C-terminal stem segment is A_q+3. In certain embodiments, the end of the N-terminal stem segment is A_p-4, and the start of the C- terminal stem segment is A_q+4. In certain embodiments, the end of the N-terminal stem segment is Ap-5, and the start of the C-terminal stem segment is A_q+5.

[00279] In certain embodiments, 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. In certain embodiments, the end of the N-terminal stem segment is A_p+2, and the start of the C-terminal stem segment is A_q-2. In certain embodiments, the end of the N-terminal stem segment is A_p+₃, and the start of the C-terminal stem segment is A_q-3. In certain embodiments, the end of the N-terminal stem segment is A_p+₄, and the start of the C- terminal stem segment is A_q-4. In certain embodiments, the end of the N-terminal stem segment is A_p+5, and the start of the C-terminal stem segment is A_q-5.

[00280] In certain embodiments, the end of the N-terminal stem segment is D_p-i, and the start of the C-terminal stem segment is D_q-i. In certain embodiments, the end of the N-terminal stem segment is D_p-2, and the start of the C-terminal stem segment is D_q-2. In certain embodiments, the end of the N-terminal stem segment is D_p-3, and the start of the C-terminal stem segment is D_q-3. In certain embodiments, the end of the N-terminal stem segment is D_p-4, and the start of the C- terminal stem segment is D_q-4. In certain embodiments, the end of the N-terminal stem segment is D_p-5, and the start of the C-terminal stem segment is D_q-5.

[00281] In certain embodiments, the end of the N-terminal stem segment is D_p+i, and the start of the C-terminal stem segment is D_q+i. In certain embodiments, the end of the N-terminal stem segment is D_p+2, and the start of the C-terminal stem segment is D_q+2. In certain embodiments, the end of the N-terminal stem segment is D_p+₃, and the start of the C-terminal stem segment is D_q+₃. In certain embodiments, the end of the N-terminal stem segment is D_p+4, and the start of the C-terminal stem segment is D_q+4. In certain embodiments, the end of the N-terminal stem segment is D_p+₅, and the start of the C-terminal stem segment is D_q+₅.

[00282] In certain embodiments, the end of the N-terminal stem segment is D_p-i, and the start of the C-terminal stem segment is D_q+i. In certain embodiments, the end of the N-terminal stem segment is D_p-2, and the start of the C-terminal stem segment is D_q+2. In certain embodiments, the end of the N-terminal stem segment is D_p-3, and the start of the C-terminal stem segment is D_q+₃. In certain embodiments, the end of the N-terminal stem segment is D_p-4, and the start of the C- terminal stem segment is D_q+4. In certain embodiments, the end of the N-terminal stem segment is D_p-5, and the start of the C-terminal stem segment is D_q+5. [00283] In certain embodiments, the end of the N-terminal stem segment is D_p+i, and the start of the C-terminal stem segment is D_q-i. In certain embodiments, the end of the N-terminal stem segment is D_p+2, and the start of the C-terminal stem segment is D_q-2. In certain embodiments, the end of the N-terminal stem segment is D_p+₃, and the start of the C-terminal stem segment is D_q-3. In certain embodiments, the end of the N-terminal stem segment is D_p+₄, and the start of the C- terminal stem segment is D_q-4. In certain embodiments, the end of the N-terminal stem segment is D_p+5, and the start of the C-terminal stem segment is D_q-5.

[00284] Also provided herein are 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. Also provided herein are influenza hemagglutinin stem domain polypeptides comprising deleted forms of a known influenza hemagglutinin stem domain, wherein 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 are deleted from the stem domain. In certain embodiments, provided herein are 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. In particular embodiments, if one residue is added to the C-terminal stem segment, then one residue is added to the N-terminal stem segment; if two residues are added to the C-terminal stem segment, then two residues are added to the N-terminal stem segment; if three residues are added to the C-terminal stem segment, then three residues are added to the N-terminal stem segment. Further provided herein are influenza hemagglutinin stem domain polypeptides comprising altered forms of HAl C-terminal stem segments 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 are conservatively substituted with other amino acids. Also provided herein are influenza hemagglutinin stem domain polypeptides comprising altered forms of HAl C- terminal stem segments, 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 HAl C-terminal stem segment are substituted (e.g., conservatively substituted) with other amino acids. Further provided are influenza hemagglutinin stem domain polypeptides comprising deleted and altered HAl C-terminal stem segments. In certain embodiments, the influenza hemagglutinin stem domain polypeptides provided herein comprise a chimeric/hybrid of the stem domain of the HAl subunit. The chimeric of the stem domain of the HAl subunit may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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 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 may be from a second influenza virus strain or subtype. In certain embodiments, 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. In certain embodiments, the influenza hemagglutinin stem domain polypeptides provided herein comprise an HA2 subunit and a chimeric of the stem domain of the HAl subunit.

[00285] The influenza hemagglutinin stem domain polypeptides might be based on (i.e. might have sequence identity, as described above) any influenza hemagglutinin known to those of skill or later discovered. In certain embodiments, influenza hemagglutinin stem domain polypeptides are based on an influenza A hemagglutinin. In certain embodiments, the influenza

hemagglutinin 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. In certain embodiments, influenza hemagglutinin stem domain polypeptides are based on an influenza B hemagglutinin, as described in detail below.

[00286] The HAl N-terminal stem segments might be based on (i.e. might have sequence identity, as described above) any HAl N-terminal stem segments known to those of skill or later discovered. In certain embodiments, the HAl N-terminal stem segments are based on influenza A HAl N-terminal stem segments. In certain embodiments, 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. In certain embodiments, 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, A/HK/4801/14, or A/Perth/16/2009 influenza virus. In certain embodiments, the HAl N-terminal stem segment is or is based on the HA-1 N-terminal stem segment of an A/California/04/2009 influenza virus.

[00287] The HAl C-terminal stem segments might be based on (i.e. might have sequence identity, as described above) any HAl C-terminal stem segments known to those of skill or later discovered. In certain embodiments, the HAl C-terminal stem segments are based on influenza A HAl C-terminal stem segments. In certain embodiments, the HAl C-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. In certain embodiments, the HAl C-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, A/HK/4801/14, or A/Perth/16/2009 influenza virus. In certain embodiments, the HAl N-terminal stem segment is or is based on the HA-1 N-terminal stem segment of an A/California/04/2009 influenza virus.

[00288] The HA2 stem domains might be based on (i.e. might have sequence identity, as described above) any HA2 stem domains known to those of skill or later discovered. In certain embodiments, the HA2 stem domains are based on influenza A HA2 stem domains. In certain embodiments, 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. In certain embodiments, the HA2 stem domain is selected from SEQ ID NOS:34-49. In certain embodiments, the HA2 stem domain is or is based on the HA stem domain of an A/ Ann Arbor/6/60-like, A/Puerto Rico/8/1934-like, A/Perth/ 16/2009-like, A/California/07/2009-like, A/HK/4801/14-like, A/Brisbane/59/07-like, A/New

Caledonia/20/1999-like or A/Victoria/361/201-like influenza virus. In certain embodiments, the HA2 stem domain is or is based on the HA stem domain of an A/California/04/2009 influenza virus or A/Perth/16/2009 influena virus. In certain embodiments, the HA2 stem domain is or is based on a later discovered HA2 stem domain.

[00289] In certain embodiments, the HA2 stem domain is from the same influenza virus strain or subtype as the stem domain of the HAl subunit.

[00290] In embodiments comprising a luminal domain, the luminal domain might be based on any influenza luminal domain known to those of skill in the art. In certain embodiments, the luminal domains are based on influenza A luminal domains. In certain embodiments, the HA2 luminal 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. In certain embodiments, the luminal domain might be any luminal domain deemed useful to one of skill in the art. In certain embodiments, the luminal domain is from the same influenza virus strain or subtype as the stem domain of the HA2 subunit.

[00291] In certain embodiments, the cytoplasmic, transmembrane and luminal domains are from the same influenza virus strain or subtype as the stem domain of the HA2 subunit. In other embodiments, the cytoplasmic and transmembrane domains are from the same influenza virus strain or subtype as the stem domain of the HA2 subunit. In certain embodiments, the cytoplasmic and luminal domain are from the same influenza virus strain or subtype as the stem domain of the HA2 subunit.

[00292] In embodiments comprising a transmembrane domain, the transmembrane domain might be based on any influenza transmembrane domain known to those of skill in the art. In certain embodiments, the transmembrane domains are based on influenza A transmembrane domains. In certain embodiments, 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, Hl l, H12, H13, H14, H15, H16, H17, and H18. In certain embodiments, the transmembrane domain might be any transmembrane domain deemed useful to one of skill in the art. In certain embodiments, the transmembrane domain is selected from SEQ ID NOS:67-82. In certain embodiments, the transmembrane domains are from the same influenza virus strain or subtype as the stem domain of the HA2 subunit.

[00293] In embodiments comprising a cytoplasmic domain, the cytoplasmic domain might be based on any influenza cytoplasmic domain known to those of skill in the art. In certain embodiments, the cytoplasmic domains are based on influenza A cytoplasmic domains. In certain embodiments, the HA2 cytoplasmic 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. In certain embodiments, the cytoplasmic domain might be any cytoplasmic domain deemed useful to one of skill in the art. In certain embodiments, the cytoplasmic domain is selected from SEQ ID NOS:83-98. In certain embodiments, the cytoplasmic domains are from the same influenza virus strain or subtype as the stem domain of the HA2 subunit. [00294] In certain embodiments, the HA stem domain is as disclosed in International

Publication Nos. WO 2011/123495, WO 2013/043729, and WO 2014/099931, U.S. Publication Nos. 2013/0129761, and 2014/0328875, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 which are incorporated herein by reference in their entirety. In certain embodiments, the HA stem domain comprises amino acid sequences as described in Tables 6, 6A, 7, and 7A of International Publication No. WO 2011/123495 and WO 2013/043729, U.S. Publication No. 2013/0129761, and U.S. Application No. 14/345,816, which published as U.S. Patent Publication No. 2015/0132330 which are incorporated by reference herein in their entirety, and Tables 1, 1A, and 2 of International Publication No. WO

2010/117786 and U.S. Publication No. 2010/0297174, which are incorporated herein by reference in their entirety.

[00295] In certain embodiments, the HA2 stem domains are based on an influenza B hemagglutinin. Exemplary residues for the end of an N-terminal stem segment and the end of a C-terminal stem segment of an influenza B hemagglutinin are indicated in Fig. 2 of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety. In certain embodiments, the HA2 stem domain is according to SEQ ID NO:99, presented in Tables 3 and 4 as disclosed in International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety.

[00296] In particular embodiments, the boundaries of the influenza B virus HA1 N-terminal stem segment and influenza B virus HA1 C-terminal segment are defined with respect to D_p and

Dq.

[00297] In other particular embodiments, the boundaries of the influenza B virus HA1 N- terminal stem segment and influenza B virus HA1 C-terminal segment are defined with respect to six pairs of amino acid residues: Argso and Ser₂77; Ala₆6 and Trp₂₇i; Lysso and Ser_277; Cys94 and Cysi4₃; Cysi78 and Cys₂72 and Cyss4 and Cys₂72. Positions of these six pairs of residues are also highlighted in Fig. 3 of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety. The residue numbers of these six pairs are based on the numbering of the B-HA from influenza virus B as described in Protein Data Bank accession No. 3BT6. The amino acid sequence corresponding to the X-ray crystal structure of the B-HA protein in Protein Data Bank accession No. 3BT6 is aligned with representative HI and H3 amino acid sequence and shown in Fig. 2 of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety. In certain embodiments, 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 Argso. In certain embodiments, an influenza B virus HAl N-terminal stem segment starts at residue 1 and ends at Ala₆6. In some embodiments, an influenza B virus HAl N-terminal stem segment starts at residue 1 and ends at Lysso. In some embodiments, an influenza B virus N-terminal stem segment starts at residue 1 and ends at Argso. 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 Cys94. In some embodiments, an influenza B virus N- terminal stem segment starts at residue 1 and ends at Cysns.

[00298] In certain embodiments, the influenza B virus HA2 domain is in tertiary or quaternary association with the influenza B virus HAl domain through the influenza B virus HAl N- terminal segment, the influenza B virus HAl C-terminal segment, or both.

[00299] In some embodiments, the influenza B virus HAl C-terminal segment and the influenza B virus HA2 subunit are covalently linked. For example, at its C-terminus (e.g., at the ending residue of the second sequence), the influenza B virus HAl C-terminal segment is covalently linked to the influenza B virus HA2 domain in such embodiments. In some embodiments, the influenza B virus HAl C-terminal segment and influenza B virus HA2 domain form a continuous polypeptide chain.

[00300] As illustrated in Fig. 14 and in Fig. 2 of International Publication No. WO

2013/043729, which is incorporated herein by reference in its entirety, HAl N-terminal stem segments share sequence identity between influenza A and influenza B and additionally across influenza A subtypes. Similarly, HAl C-terminal stem segments also share sequence identity between influenza A and influenza B and additionally across influenza A subtypes. Further, HA2 domains also share sequence identity between influenza A and influenza B and additionally across influenza A subtypes.

[00301] In some embodiments, the 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. For example, an influenza hemagglutinin stem domain polypeptide might comprise HAl N-terminal and HAl C-terminal stem segments from different influenza A virus HA subtypes. In some embodiments, the HAl N-terminal stem segment is from influenza A virus while the HAl C-terminal stem segment is from influenza B virus.

Similarly, HA2 may also be from influenza A virus while the HAl N-terminal and/or C-terminal stem segment is from influenza B virus.

[00302] It will be understood that any combination of the sequence elements listed in Tables 1-4 of International Publication No. WO 2013/043729, which is incorporated herein in its entirety, or the variants thereof may be used to form the hemagglutinin HA stem domain polypeptides described herein.

[00303] In certain embodiments, 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

[DOI: 10.1126/science. l l71491], and Kashyap et al, 2008, Proc. Natl. Acad. Sci. USA

105(16):5986-5991, the contents of which are hereby incorporated by reference in their entireties. In certain embodiments, 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.

[00304] In certain embodiments, structural similarity might be assessed by spectroscopic techniques such as circular dichroism, Raman spectroscopy, NMR, 3D NMR and X-ray crystallography. Known influenza hemagglutinin structures determined by X-ray

crystallography are described in structural coordinates in Protein Data Bank files including but not limited to 1HGJ (an HA H3N2 strain) and 1RUZ (an HA H1N1 strain).

[00305] In certain embodiments, structural similarity is evaluated by RMS deviation between corresponding superimposed portions of two structures. In order to create a meaningful superimposition, in certain embodiments, the coordinates of at least 20 corresponding atoms, 25 corresponding atoms, 30 corresponding atoms, 40 corresponding atoms, 50 corresponding atoms, 60 corresponding atoms, 70 corresponding atoms, 80 corresponding atoms, 90 corresponding atoms, 100 corresponding atoms, 120 corresponding atoms, 150 corresponding atoms, 200 corresponding atoms, or 250 corresponding atoms are used to calculate an RMS deviation.

[00306] In certain embodiments, the coordinates of all corresponding atoms in amino acid backbones are used to calculate an RMS deviation. In certain embodiments, the coordinates of all corresponding alpha carbon-atoms in the amino acid backbones are used to calculate an RMS deviation. In certain embodiments, the coordinates of all corresponding identical residues, including side chains, are used to calculate an RMS deviation.

[00307] In certain embodiments, coordinates of all or a portion of the corresponding atoms in a HAl N-terminal segment are used to calculate an RMS deviation. In certain embodiments, coordinates of all or a portion of the corresponding atoms in a HAl C-terminal segment are used to calculate an RMS deviation. In certain embodiments, coordinates of all or a portion of the corresponding atoms in both a HAl N-terminal segment and a C-terminal segment are used to calculate an RMS deviation. In certain embodiments, coordinates of all or a portion of corresponding atoms in HA2 domains are used to calculate an RMS deviation.

[00308] In certain embodiments, 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 (e.g., from 1HGJ or 1RUZ) 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

(Cornell University), and SuperPose (University of Alberta, Edmonton).

[00309] In certain embodiments, provided herein are influenza virus hemagglutinin stem domain polypeptides comprising a modified multi-basic cleavage site. In a specific embodiment, an influenza virus stem domain polypeptide described herein does not contain a multi-basic cleavage site.

[00310] In certain embodiments, provided herein are influenza hemagglutinin stem domain polypeptides that are predicted to be resistant to protease cleavage at the junction between HAl and HA2. Those of skill in the art should recognize that the Arg-Gly sequence spanning HAl and HA2 is a recognition site for trypsin and is typically cleaved for hemagglutinin activation. Since the stem domain polypeptides described herein need not be activated, provided herein are influenza hemagglutinin stem domain polypeptides that are predicted to be resistant to protease cleavage. In certain embodiments, provided is any influenza hemagglutinin 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 stem domain polypeptide described herein wherein the C-terminal residue of the HAl C-terminal stem segment is any residue other than Lys or Arg. In certain embodiments, provided is any influenza hemagglutinin stem domain polypeptide described herein wherein the N-terminal residue of the HA2 domain is proline. In certain embodiments, provided is any influenza hemagglutinin stem domain polypeptide described herein wherein the C-terminal residue of the HAl C-terminal stem segment is Ala and the N-terminal residue of the HA2 domain is also Ala.

5.4 NUCLEIC ACID SEQUENCES ENCODING CHIMERIC HEMAGGLUTININ

( Ι ΙΛ) POLYPEPTIDES

[00311] Provided herein are nucleic acid secquences that encode the chimeric influenza virus hemagglutinin polypeptides described herein. Due to the degeneracy of the genetic code, any nucleic acid sequence that encodes a chimeric hemagglutinin (HA) polypeptide described herein is encompassed herein. In certain embodiments, nucleic acid sequences corresponding to naturally occurring influenza virus nucleic acid sequences encoding an HAl N-terminal stem segment, an HAl C-terminal stem segment, HA2 domain, HA stem domain, HA luminal domain, HA transmembrane domain, and/or HA cytoplasmic domain are used to produce a chimeric influenza virus hemagglutinin polypeptide. In certain embodiments, nucleic acid sequences corresponding to naturally occurring influenza virus nucleic acid sequences encoding an HAl domain (e.g., an HAl stem segment (such as, an HAl N-terminal stem segment and an HAl C-terminal stem segment)), HA2 domain, HA stem domain, HA luminal domain, HA transmembrane domain, and/or HA cytoplasmic domain are used to produce a chimeric influenza virus hemagglutinin polypeptide. In certain embodiments, the nucleic acid sequence comprises one, two, or three of the following: a nucleotide sequence encoding an influenza virus HA signal peptide, a nucleotide sequence encoding an influenza virus HA transmembrane domain, and a nucleotide sequence encoding an influenza vims HA cytoplasmic domain. In specific embodiments, the nucleic acid sequence comprises nucleotide sequences encoding said chimeric HA polypeptide and preferably comprises the 5' non-coding region and 3' non-coding region from the HA of the same influenza virus as the influenza virus engineered to express the chimeric HA polypeptide. In specific embodiments, the nucleic acid sequence comprises nucleotide sequences encoding said chimeric HA polypeptide, and the 5' non-coding region, 3' non-coding region, and nucleotide sequences encoding the signal peptide from the HA of the same influenza virus as the influenza virus engineered to express the chimeric HA polypeptide. In specific embodiments, the nucleic acid sequence comprises nucleotide sequences encoding said chimeric HA polypeptide, and the 5' non-coding region and 3' non-coding region from the HA of the same influenza virus as the influenza virus engineered to express the chimeric HA polypeptide, and nucleotide sequences encoding one, two, or three of the following: the influenza virus HA signal peptide, the influenza virus HA transmembrane domain, and the influenza virus HA cytoplasmic domain from the HA of the same influenza virus as the influenza virus engineered to express the chimeric HA polypeptide.

[00312] Also provided herein are nucleic acid sequences capable of hybridizing to a nucleic acid encoding a chimeric influenza virus hemagglutinin polypeptide. In certain embodiments, provided herein are nucleic acid sequences capable of hybridizing to a fragment of a nucleic acid sequence encoding a chimeric influenza virus hemagglutinin polypeptide. In other

embodiments, provided herein are nucleic acid sequences capable of hybridizing to the full length of a nucleic acid sequence encoding a chimeric influenza virus hemagglutinin

polypeptide. General parameters for hybridization conditions for nucleic acids are described in Sambrook et al, Molecular Cloning - A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), and in Ausubel et al, Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New York (1994).

Hybridization may be performed under high stringency conditions, medium stringency conditions, or low stringency conditions. Those of skill in the art will understand that low, medium and high stringency conditions are contingent upon multiple factors all of which interact and are also dependent upon the nucleic acids in question. For example, high stringency conditions may include temperatures within 5°C melting temperature of the nucleic acid(s), a low salt concentration {e.g., less than 250 mM), and a high co-solvent concentration {e.g., 1-20% of co-solvent, e.g., DMSO). Low stringency conditions, on the other hand, may include temperatures greater than 10°C below the melting temperature of the nucleic acid(s), a high salt concentration (e.g., greater than 1000 mM) and the absence of co-solvents. In specific embodiments, the nucleic acid sequence is capable of hybridizing under high stringency conditions to the full length of the nucleotide sequence set forth in SEQ ID NOs: 140, 184, 186, 188.. In specific embodiments, the nucleic acid sequence is capable of hybridizing under high stringency conditions to the full length of the coding sequence of the nucleotide sequence set forth in SEQ ID NO: 140, 184, 186, 188. In a specific embodiment, a chimeric HA is encoded by the nucleic acid sequence set forth in Fig. 28, 30, 32, or 34 or the complement thereof. In a specific embodiment, a chimeric HA is encoded by a nucleic acid sequence comprising the nucleotide sequence encoding the ectodomain set forth in Fig. 28, 30, 32, or 34 (i.e., excluding the 5' and 3' noncoding regions and nucleic acid sequences encoding the signal peptide, transmembrane domain, and cytoplasmic domain set forth in Fig. 28, 30, 32, or 34, respectively), or the complement thereof.

[00313] In a specific embodiment, a chimeric HA polypeptide is encoded by a nucleic acid sequence comprising the nucleotide sequence encoding the ectodomain set forth in FIG. 28, 30, 32, or 34, or a complement thereof, and one, two or all of the following: (1) the nucleotide sequence encoding the signal peptide set forth in FIG. 28, 30, 32, or 34, respectively, or a complement thereof; (2) the nucleotide sequence encoding the transmembrane domain set forth in FIG. 28, 30, 32, or 34, respectively, or a complement thereof; and (3) the nucleotide sequence encoding the cytoplasmic domain set forth in FIG. 28, 30, 32, or 34, respectively, or a complement thereof.

[00314] In a specific embodiment, a nucleic acid sequence encoding a chimeric HA polypeptide comprises the nucleotide sequence set forth in FIG. 28, 30, 32, or 34, or a complement thereof. In another specific embodiment, a nucleic acid sequence encoding a chimeric HA polypeptide comprises the nucleotide sequence set forth in FIG. 28, 30, 32, or 34, or a complement thereof, without the signal peptide. In another specific embodiment, a nucleic acid sequence encoding a chimeric HA polypeptide comprises the nucleotide sequence set forth in FIG. 28, 30, 32, or 34, or a complement thereof, without the 5' non-coding region, 3' non- coding region or both. In another specific embodiment, a nucleic acid sequence encoding a chimeric HA polypeptide comprises the nucleotide sequence set forth in FIG. 28, 30, 32, or 34, or a complement thereof, without the signal peptide and without the 5' non-coding region, 3' non-coding region or both.

[00315] In some embodiments, a nucleic acid sequence encoding a chimeric influenza virus hemagglutinin polypeptide is isolated. In certain embodiments, an "isolated" nucleic acid sequence refers to a nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. In other words, the isolated nucleic acid sequence can comprise heterologous nucleic acids that are not associated with it in nature. In other embodiments, an "isolated" nucleic acid sequence, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of nucleic acid sequences in which the nucleic acid sequence is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, nucleic acid sequence that is substantially free of cellular material includes preparations of nucleic acid sequence having less than about 30%, 20%, 10%, or 5% (by dry weight) of other nucleic acids. The term

"substantially free of culture medium" includes preparations of nucleic acid sequence in which the culture medium represents less than about 50%, 20%, 10%, or 5% of the volume of the preparation. The term "substantially free of chemical precursors or other chemicals" includes preparations in which the nucleic acid sequence is separated from chemical precursors or other chemicals which are involved in the synthesis of the nucleic acid sequence. In specific embodiments, such preparations of the nucleic acid sequence have less than about 50%, 30%, 20%), 10%), 5% (by dry weight) of chemical precursors or compounds other than the nucleic acid sequence of interest.

[00316] In addition, provided herein are nucleic acid sequences encoding the individual components of a chimeric influenza virus hemagglutinin polypeptide. In specific embodiments, nucleic acid sequences encoding the globular head domain and/or the stem domain of the chimeric influenza virus hemagglutinin polypeptide are provided. Nucleic acid sequences encoding components of a chimeric influenza virus hemagglutinin polypeptide may be assembled using standard molecular biology techniques known to one of skill in the art. In specific embodiments, the individual components of a chimeric influenza virus hemagglutinin polypeptide can be expressed by the same or different vector. 5.5 EXPRESSION OF A CHIMERIC HEMAGGLUTININ (HA) POLYPEPTIDE

[00317] Provided herein are vectors, including expression vectors, containing a nucleic acid sequence encoding a chimeric influenza virus hemagglutinin polypeptide described herein. In a specific embodiment, the vector is an expression vector that is capable of directing the expression of a nucleic acid sequence encoding a chimeric influenza virus hemagglutinin polypeptide. Non-limiting examples of expression vectors include, but are not limited to, plasmids and viral vectors, such as replication defective retroviruses, adenoviruses, adeno- associated viruses and baculoviruses. Expression vectors also may include, without limitation, transgenic animals and non-mammalian cells/organisms, e.g., mammalian cells/organisms that have been engineered to perform mammalian N-linked glycosylation.

[00318] In some embodiments, provided herein are expression vectors encoding components of a chimeric hemagglutinin (HA) polypeptide (e.g., the stem domain and the head domain, or portions of either domain). Such vectors may be used to express the components in one or more host cells and the components may be isolated and conjugated together with a linker using techniques known to one of skill in the art.

[00319] An expression vector comprises a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide described herein and in a form suitable for expression of the nucleic acid sequence in a host cell. In a specific embodiment, an expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid to be expressed. Within an expression vector, "operably linked" is intended to mean that a nucleic acid of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleic acid sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleic acid in many types of host cells, those which direct expression of the nucleic acid only in certain host cells (e.g., tissue-specific regulatory sequences), and those which direct the expression of the nucleic acid upon stimulation with a particular agent (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The term "host cell" is intended to include a particular subject cell transformed or transfected with a nucleic acid sequence and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transformed or transfected with the nucleic acid sequence due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid sequence into the host cell genome. In specific embodiments, the host cell is a cell line.

[00320] Expression vectors can be designed for expression of a chimeric hemagglutinin (HA) polypeptide described herein using prokaryotic (e g, E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors, see, e.g., Treanor et al, 2007, JAMA, 297(14): 1577-1582 incorporated by reference herein in its entirety), yeast cells, plant cells, algae, avian, or mammalian cells). Examples of yeast host cells include, but are not limited to S. pombe and S. cerevisiae and examples, infra. An example of avian cells includes, but is not limited to EB66 cells. Examples of mammalian host cells include, but are not limited to, Crucell Per.C6 cells, Vero cells, CHO cells, VERO cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293 cells, 3T3 cells or WI38 cells. In certain embodiments, the hosts cells are myeloma cells, e.g., NS0 cells, 45.6 TGI .7 cells, AF-2 clone 9B5 cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells, NCI-H929 cells, P cells, NSO/1 cells, P3 NS1 Ag4 cells, P3/NSl/l-Ag4-l cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U. l cells, RPMI 8226 cells, Sp20-Agl4 cells, U266B1 cells, X63AG8.653 cells, Y3.Ag.1.2.3 cells, and YO cells. Non- limiting examples of insect cells include 5/9, Sfll, Trichoplusia ni, Spodoptera frugiperda and Bombyx mori. In a particular embodiment, a mammalian cell culture system (e.g. Chinese hamster ovary or baby hamster kidney cells) is used for expression of a chimeric hemagglutinin (HA) polypeptide. In another embodiment, a plant cell culture system is used for expression of a chimeric hemagglutinin (HA) polypeptide. See, e.g., U.S. Patent Nos. 7,504,560; 6,770,799; 6,551,820; 6, 136,320; 6,034,298; 5,914,935; 5,612,487; and 5,484,719, and U.S. patent application publication Nos. 2009/0208477, 2009/0082548, 2009/0053762, 2008/0038232, 2007/0275014 and 2006/0204487 for plant cells and methods for the production of proteins utilizing plant cell culture systems. In specific embodiments, plant cell culture systems are not used for expression of a chimeric hemagglutinin (HA) polypeptide. The host cells comprising the nucleic acids that encode the chimeric hemagglutinin (HA) polypeptides described herein can be isolated, i.e., the cells are outside of the body of a subject. In certain embodiments, the cells are engineered to express nucleic acids that encode the chimeric influenza virus hemagglutinin polypeptides described herein. In specific embodiments, the host cells are cells from a cell line.

[00321] In a specific embodiment, provided herein are host cells comprising a nucleic acid sequence comprising a nucleotide sequence encoding a chimeric influenza virus HA polypeptide described herein. In a specific embodiment, provided herein are host cells engineered to express a nucleic acid sequence comprising a nucleotide sequence encoding a chimeric influenza virus HA polypeptide described herein. In a specific embodiment, provided herein are host cells comprising an expression vector comprising nucleic acid sequence encoding a chimeric influenza virus HA polypeptide described herein. Host cells are known to one of skill in the art and examples are provided herein. In specific embodiments, the host cells are cells from a cell line.

[00322] An expression vector can be introduced into host cells via conventional

transformation or transfection techniques. Such techniques include, but are not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al, 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, and other laboratory manuals. In certain embodiments, a host cell is transiently transfected with an expression vector containing a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide. In other embodiments, a host cell is stably transfected with an expression vector containing a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide.

[00323] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a nucleic acid that encodes a selectable marker {e.g., for resistance to antibiotics) is generally introduced into the host cells along with the nucleic acid of interest. Examples of selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid sequence can be identified by drug selection {e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). [00324] As an alternative to recombinant expression of a chimeric hemagglutinin (HA) polypeptide using a host cell, an expression vector containing a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide can be transcribed and translated in vitro using, e.g., T7 promoter regulatory sequences and T7 polymerase. In a specific embodiment, a coupled transcription/translation system, such as Promega TNT®, or a cell lysate or cell extract comprising the components necessary for transcription and translation may be used to produce a chimeric hemagglutinin (HA) polypeptide.

[00325] Once a chimeric hemagglutinin (HA) polypeptide has been produced, it may be isolated or purified by any method known in the art for isolation or purification of a protein, for example, by chromatography {e.g., ion exchange, affinity, particularly by affinity for the specific antigen, by Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the isolation or purification of proteins.

[00326] Accordingly, provided herein are methods for producing a chimeric influenza virus hemagglutinin (HA) polypeptide. In one embodiment, the method comprises culturing a host cell containing a nucleic acid sequence encoding the polypeptide in a suitable medium such that the polypeptide is produced. In a specific embodiment, the method comprises culturing a host cell containing a nucleic acid sequence comprising a nucleotide sequence encoding the polypeptide, or a host cell containing an expression vector containing a nucleic acid sequence comprising a nucleotide sequence encoding the polypeptide in a suitable medium such that the polypeptide is produced. In some embodiments, the method further comprises isolating the polypeptide from the medium or the host cell.

[00327] Also provided herein are methods for producing a virus comprising a chimeric HA described herein, comprising propagating the virus in any substrate that allows the virus to grow to titers that permit their use in accordance with the methods described herein. In one embodiment, the substrate allows the viruses to grow to titers comparable to those determined for the corresponding wild-type viruses. In a specific embodiment, the virus is propagated in embryonated eggs {e.g., chicken eggs). In a specific embodiment, the virus is propagated in 8 day old, 9-day old, 8-10 day old, 10 day old, 11-day old, 10-12 day old, or 12-day old

embryonated eggs {e.g., chicken eggs). In certain embodiments, the virus is propagated in MDCK cells, Vero cells, 293T cells, or other cell lines known in the art. In certain

embodiments, the virus is propagated in cells derived from embryonated eggs. 5.6 INFLUENZA VIRUS VECTORS

[00328] In one aspect, provided herein are influenza viruses containing a chimeric influenza virus hemagglutinin polypeptide described herein. In a specific embodiment, the chimeric hemagglutinin (HA) polypeptide is incorporated into the virions of the influenza virus. The influenza viruses may be conjugated to moieties that target the viruses to particular cell types, such as immune cells. In some embodiments, the virions of the influenza virus have

incorporated into them or express a heterologous polypeptide in addition to a chimeric hemagglutinin (HA) polypeptide. The heterologous polypeptide may be a polypeptide that has immunopotentiating activity, or that targets the influenza virus to a particular cell type, such as an antibody that binds to an antigen on a specific cell type or a ligand that binds a specific receptor on a specific cell type.

[00329] Influenza viruses containing a chimeric hemagglutinin (HA) polypeptide may be produced by supplying in trans the chimeric hemagglutinin (HA) polypeptide during production of virions using techniques known to one skilled in the art, such as reverse genetics and helper- free plasmid rescue. Alternatively, the replication of a parental influenza virus comprising a genome engineered to express a chimeric hemagglutinin (HA) polypeptide in cells susceptible to infection with the virus wherein hemagglutinin function is provided in trans will produce progeny influenza viruses containing the chimeric hemagglutinin (HA) polypeptide.

[00330] In another aspect, provided herein are influenza viruses comprising a genome engineered to express a chimeric hemagglutinin (HA) polypeptide. In a specific embodiment, the genome of a parental influenza virus is engineered to encode a chimeric hemagglutinin (HA) polypeptide, which is expressed by progeny influenza virus. In another specific embodiment, the genome of a parental influenza virus is engineered to encode a chimeric hemagglutinin (HA) polypeptide, which is expressed and incorporated into the virions of progeny influenza virus. Thus, the progeny influenza virus resulting from the replication of the parental influenza virus contain a chimeric hemagglutinin (HA) polypeptide. The virions of the parental influenza virus may have incorporated into them a chimeric hemagglutinin (HA) polypeptide that contains a stem or head domain from the same or a different type, subtype/lineage or strain of influenza virus. Alternatively, the virions of the parental influenza virus may have incorporated into them a moiety that is capable of functionally replacing one or more of the activities of influenza virus hemagglutinin polypeptide {e.g., the receptor binding and/or fusogenic activities of influenza virus hemagglutinin). In specific embodiments, the parental influenza virus is an influenza A virus. In specific embodiments, the parental influenza virus is an influenza B virus.

[00331] In some embodiments, the virions of the parental influenza virus have incorporated into them a heterologous polypeptide. In certain embodiments, the genome of a parental influenza virus is engineered to encode a heterologous polypeptide and a chimeric hemagglutinin (HA) polypeptide, which are expressed by progeny influenza virus. In specific embodiments, the chimeric hemagglutinin (HA) polypeptide, the heterologous polypeptide or both are incorporated into virions of the progeny influenza virus.

[00332] In some embodiments, the virions of the parental influenza virus have incorporated into them an influenza virus neuraminidase, wherein the strain of the influenza virus

neuraminidase is not the same strain as the strain of the globular head domain or stem domain of the chimeric influenza virus HA polypeptide. In some embodiments, the influenza virus neuraminidase corresponds to the influenza virus neuraminidase of the HA stem domain of the chimeric influenza virus HA polypeptide. In some embodiments, the influenza virus

neuraminidase corresponds to the influenza virus neuraminidase of the HA globular head domain of the chimeric influenza virus HA polypeptide.

[00333] Since the genome of influenza A and B viruses consist of eight (8) single-stranded, negative sense segments (influenza C viruses consist of seven (7) single-stranded, negative sense segments), the genome of a parental influenza virus may be engineered to express a chimeric hemagglutinin (HA) polypeptide (and any other polypeptide, such as a heterologous polypeptide) using a recombinant segment and techniques known to one skilled in the art, such a reverse genetics and helper-free plasmid rescue. In one embodiment, the recombinant segment comprises a nucleic acid encoding the chimeric hemagglutinin (HA) polypeptide as well as the 3' and 5' incorporation signals which are required for proper replication, transcription and packaging of the vRNAs (Fujii et al, 2003, Proc. Natl. Acad. Sci. USA 100:2002-2007; Zheng, et al., 1996, Virology 217:242-251, International Publication No. WO 2011/014645, all of which are incorporated by reference herein in their entireties). In a specific embodiment, the recombinant segment uses the 3' and 5' noncoding and/or nontranslated sequences of segments of influenza viruses that are from a different or the same type, subtype/lineage or strain as the parental influenza virus. In some embodiments, the recombinant segment comprises the 3' noncoding region of an influenza virus hemagglutinin polypeptide, the untranslated regions of an influenza vims hemagglutinin polypeptide, and the 5' non-coding region of an influenza vims hemagglutinin polypeptide. In specific embodiments, the recombinant segment comprises the 3' and 5' noncoding and/or nontranslated sequences of the HA segment of an influenza vims that is the same type, subtype/lineage or strain as the influenza vims type, subtype/lineage or strain as the HAl N-terminal stem segment, the HAl C-terminal stem segment, the globular head domain, and/or the HA2 of a chimeric hemagglutinin (HA) polypeptide. In specific embodiments, the recombinant segment comprises packaging signals, such as the 5' and 3' non-coding regions and signal peptide of the HA segment of an influenza vims, from the same type, lineage, or strain as the influenza vims backbone. For example, if the chimeric HA is engineered to be expressed from an influenza A vims, then the nucleotide sequence encoding chimeric HA comprises the 5' and 3' non-coding regions and the nucleotide sequence encoding the signal peptide of the HA segment of the influenza A vims. In another example, if the chimeric HA is engineered to be expressed from an influenza B vims, then the nucleotide sequence encoding chimeric HA comprises the 5' and 3' non-coding regions and the nucleotide sequence encoding the signal peptide of the HA segment of the influenza B vims. In certain embodiments, the recombinant segment encoding the chimeric hemagglutinin (HA) polypeptide may replace the HA segment of a parental influenza vims.

[00334] In some embodiments, a chimeric hemagglutinin gene segment encodes a chimeric hemagglutinin (HA) polypeptide. In specific embodiments, the chimeric hemagglutinin (HA) gene segment and at least one other influenza vims gene segment comprise packaging signals that enable the chimeric hemagglutinin (HA) gene segment and the at least one other gene segment to segregate together during replication of a recombinant influenza vims (see, Gao & Palese 2009, PNAS 106: 15891-15896; U.S. Patent No. 8,828,406; and International Application Publication No. WOl 1/014645).

[00335] In some embodiments, the genome of a parental influenza vims may be engineered to express a chimeric hemagglutinin (HA) polypeptide using a recombinant segment that is bicistronic. Bicistronic techniques allow the engineering of coding sequences of multiple proteins into a single mRNA through the use of internal ribosome entry site (IRES) sequences. IRES sequences direct the internal recmitment of ribosomes to the RNA molecule and allow downstream translation in a cap independent manner. Briefly, a coding region of one protein is inserted into the open reading frame (ORF) of a second protein. The insertion is flanked by an IRES and any untranslated signal sequences necessary for proper expression and/or function. The insertion must not disrupt the ORF, polyadenylation or transcriptional promoters of the second protein (see, e.g., Garcia-Sastre et al, 1994, J. Virol. 68:6254-6261 and Garcia-Sastre et al, 1994 Dev. Biol. Stand. 82:237-246, each of which is hereby incorporated by reference in its entirety). See also, e.g., U.S. Patent No. 6,887,699, U.S. Patent No. 6,001,634, U.S. Patent No. 5,854,037 and U.S. Patent No. 5,820,871, each of which is incorporated herein by reference in its entirety. Any IRES known in the art or described herein may be used in accordance with the invention {e.g., the IRES of BiP gene, nucleotides 372 to 592 of GenBank database entry HUMGRP78; or the IRES of encephalomyocarditis virus (EMCV), nucleotides 1430-2115 of GenBank database entry CQ867238.). Thus, in certain embodiments, a parental influenza virus is engineered to contain a bicistronic RNA segment that expresses the chimeric hemagglutinin (HA) polypeptide and another polypeptide, such as a gene expressed by the parental influenza virus. In some embodiments, the parental influenza virus gene is the HA gene

[00336] Techniques known to one skilled in the art may be used to produce an influenza virus containing a chimeric hemagglutinin (HA) polypeptide and an influenza virus comprising a genome engineered to express a chimeric hemagglutinin (HA) polypeptide. For example, reverse genetics techniques may be used to generate such an influenza virus. Briefly, reverse genetics techniques generally involve the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the negative-strand, viral RNA which are essential for the recognition by viral polymerases and for packaging signals necessary to generate a mature virion. The recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant

ribonucleoproteins (RNPs) which can be used to transfect cells. A more efficient transfection is achieved if the viral polymerase proteins are present during transcription of the synthetic RNAs either in vitro or in vivo. The synthetic recombinant RNPs can be rescued into infectious virus particles. The foregoing techniques are described in U.S. Patent No. 5, 166,057 issued November 24, 1992; in U.S. Patent No. 5,854,037 issued December 29, 1998; in European Patent

Publication EP 0702085A1, published February 20, 1996; in U.S. Patent Application Serial No. 09/152,845; in International Patent Publications PCT WO 97/12032 published April 3, 1997; WO 96/34625 published November 7, 1996; in European Patent Publication EP A780475; WO 99/02657 published January 21, 1999; WO 98/53078 published November 26, 1998; WO 98/02530 published January 22, 1998; WO 99/15672 published April 1, 1999; WO 98/13501 published April 2, 1998; WO 97/06270 published February 20, 1997; and EPO 780 475 Al published June 25, 1997, each of which is incorporated by reference herein in its entirety.

[00337] Alternatively, helper-free plasmid technology may be used to produce an influenza virus containing a chimeric hemagglutinin (HA) polypeptide and an influenza virus comprising a genome engineered to express a chimeric hemagglutinin (HA) polypeptide. Briefly, full length cDNAs of viral segments are amplified using PCR with primers that include unique restriction sites, which allow the insertion of the PCR product into the plasmid vector (Flandorfer et al, 2003, J. Virol. 77:9116-9123; Nakaya et a/., 2001, J. Virol. 75: 11868-11873; both of which are incorporated herein by reference in their entireties). The plasmid vector is designed so that an exact negative (vRNA sense) transcript is expressed. For example, the plasmid vector may be designed to position the PCR product between a truncated human RNA polymerase I promoter and a hepatitis delta virus ribozyme sequence such that an exact negative (vRNA sense) transcript is produced from the polymerase I promoter. Separate plasmid vectors comprising each viral segment as well as expression vectors comprising necessary viral proteins may be transfected into cells leading to production of recombinant viral particles. In another example, plasmid vectors from which both the viral genomic RNA and mRNA encoding the necessary viral proteins are expressed may be used. For a detailed description of helper-free plasmid technology see, e.g., International Publication No. WO 01/04333; U.S. Patent Nos. 6,951,754, 7,384,774, 6,649,372, and 7,312,064; Fodor et a/., 1999, J. Virol. 73 :9679-9682; Quinlivan et a/., 2005, J. Virol. 79:8431-8439; Hoffmann et a/., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113; and Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96:9345-9350, each of which is incorporated herein by reference in its entirety.

[00338] The influenza viruses described herein may be propagated in any substrate that allows the virus to grow to titers that permit their use in accordance with the methods described herein. Thus, in certain embodiments, provided herein is a method for producing a virus described herein comprising propagating the virus in a substrate. In one embodiment, the substrate allows the viruses to grow to titers comparable to those determined for the corresponding wild-type viruses. In certain embodiments, the substrate is one which is biologically relevant to the influenza virus or to the virus from which the HA function is derived. In a specific embodiment, an attenuated influenza virus by virtue of, e.g., a mutation in the NS1 gene, may be propagated in an IFN-deficient substrate. For example, a suitable IFN-deficient substrate may be one that is defective in its ability to produce or respond to interferon, or is one which an IFN-deficient substrate may be used for the growth of any number of viruses which may require interferon- deficient growth environment. See, for example, U.S. Patent Nos. 6,573,079, issued June 3, 2003, 6,852,522, issued February 8, 2005, and 7,494,808, issued February 24, 2009, the entire contents of each of which is incorporated herein by reference in its entirety. In a specific embodiment, the virus is propagated in embryonated eggs {e.g., chicken eggs). In a specific embodiment, the virus is propagated in 8 day old, 9-day old, 8-10 day old, 10 day old, 11-day old, 10-12 day old, or 12-day old embryonated eggs {e.g., chicken eggs). In certain

embodiments, the virus is propagated in a cell line susceptible to influenza virus infection. In certain embodiments, the virus is propagated in MDCK cells, Vero cells, 293T cells, or other cell lines known in the art. In certain embodiments, the virus is propagated in cells derived from embryonated eggs.

[00339] The influenza viruses described herein may be isolated and purified by any method known to those of skill in the art. In one embodiment, the virus is removed from cell culture and separated from cellular components, typically by well known clarification procedures, e.g., such as gradient centrifugation and column chromatography, and may be further purified as desired using procedures well known to those skilled in the art, e.g., plaque assays.

[00340] In certain embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from an influenza A virus. In certain embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from a single influenza A virus subtype/lineage or strain. In other embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from two or more influenza A virus subtypes or strains. In a specific embodiment, the influenza A virus is an influenza virus of the HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18 subtype. In a specific embodiment, the influenza A virus is an influenza virus of the H2, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or HI 8 subtype. In a specific embodiment, the influenza A virus is an influenza virus of the H5, H8, HI 1, H12, or H13 subtype. In a specific embodiment, the influenza A virus is an influenza virus of the H5 subtype. In a specific embodiment, the influenza A virus is an influenza virus of the H8 subtype. In a specific embodiment, the influenza A virus is an influenza virus of the HI 1 subtype. In a specific embodiment, the influenza A virus is an influenza virus of the H12 subtype. In a specific embodiment, the influenza A virus is an influenza virus of the H13 subtype. In a specific embodiment, the influenza A virus is an avian influenza virus.

[00341] In some embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from an influenza B virus. In certain embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from a single influenza B virus lineage or strain. In other embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from two or more influenza B virus lineages or strains. In other embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from a combination of influenza A and influenza B virus lineages or strains.

[00342] In some embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from an influenza C virus. In certain embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from a single influenza C virus subtype or strain. In other embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from two or more influenza C virus subtypes or strains. In other embodiments, the influenza viruses, or influenza virus polypeptides, genes or genome segments for use as described herein are obtained or derived from a combination of influenza C virus and influenza A virus and/or influenza B virus subtypes or strains.

[00343] Non-limiting examples of influenza A viruses include subtype H10N4, subtype H10N5, subtype H10N7, subtype H10N8, subtype H10N9, subtype HI 1N1, subtype HI 1N13, subtype HI 1N2, subtype HI 1N4, subtype HI 1N6, subtype HI 1N8, subtype HI 1N9, subtype H12N1, subtype H12N4, subtype H12N5, subtype H12N8, subtype H13N2, subtype H13N3, subtype H13N6, subtype H13N7, subtype H14N5, subtype H14N6, subtype H15N8, subtype H15N9, subtype H16N3, subtype HlNl, subtype H1N2, subtype H1N3, subtype H1N6, subtype H1N9, subtype H2N1, subtype H2N2, subtype H2N3, subtype H2N5, subtype H2N7, subtype H2N8, subtype H2N9, subtype H3N1, subtype H3N2, subtype H3N3, subtype H3N4, subtype H3N5, subtype H3N6, subtype H3N8, subtype H3N9, subtype H4N1, subtype H4N2, subtype H4N3, subtype H4N4, subtype H4N5, subtype H4N6, subtype H4N8, subtype H4N9, subtype H5N1, subtype H5N2, subtype H5N3, subtype H5N4, subtype H5N6, subtype H5N7, subtype H5N8, subtype H5N9, subtype H6N1, subtype H6N2, subtype H6N3, subtype H6N4, subtype H6N5, subtype H6N6, subtype H6N7, subtype H6N8, subtype H6N9, subtype H7N1, subtype H7N2, subtype H7N3, subtype H7N4, subtype H7N5, subtype H7N7, subtype H7N8, subtype H7N9, subtype H8N4, subtype H8N5, subtype H9N1, subtype H9N2, subtype H9N3, subtype H9N5, subtype H9N6, subtype H9N7, subtype H9N8, and subtype H9N9.

[00344] Specific examples of strains of influenza A virus include, but are not limited to: A/Victoria/361/2011 (H3N2); A/California/4/2009 (HlNl); A/California/7/2009 (HlNl);

A/Perth/16/2009 (H3N2); A/Brisbane/59/2007 (HlNl); A/Brisbane/10/2007 ((H3N2);

A/sw/Iowa/15/30 (HlNl); A/WSN/33 (HlNl); A/eq/Prague/1/56 (H7N7); A/PR/8/34;

A/mallard/Potsdam/178-4/83 (H2N2); A/herring gull/DE/712/88 (H16N3); A/sw/Hong

Kong/168/1993 (HlNl); A/mallard/Alberta/211/98 (HlNl); A/shorebird/Delaware/168/06 (H16N3); A/sw/Netherlands/25/80 (HlNl); A/sw/Germany/2/81 (HlNl); A/sw/Hannover/1/81 (HlNl); A/sw/Potsdam/1/81 (HlNl); A/sw/Potsdam/ 15/81 (HlNl); A/sw/Potsdam/268/81 (HlNl); A/sw/Finistere/2899/82 (HlNl); A/sw/Potsdam/35/82 (H3N2); A/sw/Cote

d'Armor/3633/84 (H3N2); A/sw/Gent/1/84 (H3N2); A/sw/Netherlands/12/85 (HlNl);

A/sw/Karrenzien/2/87 (H3N2); A/sw/Schwerin/103/89 (HlNl); A/turkey/Germany/3/91 (HlNl); A/sw/Germany/8533/91 (HlNl); A/sw/Belgium/220/92 (H3N2); A/sw/Gent/V230/92 (HlNl); A/sw/Leipzig/145/92 (H3N2); A/sw/Re220/92hp (H3N2); A/sw/Bakum/909/93 (H3N2); A/sw/Schleswig-Holstein/1/93 (HlNl); A/sw/Scotland/419440/94 (H1N2);

A/sw/Bakum/5/95 (HlNl); A/sw/B est/5 C/96 (HlNl); A/sw/England/17394/96 (H1N2);

A/sw/Jena/5/96 (H3N2); A/sw/Oedenrode/7C/96 (H3N2); A/sw/Lohne/1/97 (H3N2); A/sw/Cote d'Armor/790/97 (H1N2); A/sw/B akum/ 1362/98 (H3N2); A/sw/Italy/1521/98 (H1N2);

A/sw/Italy/1553-2/98 (H3N2); A/sw/Italy/1566/98 (HlNl); A/sw/Italy/ 1589/98 (HlNl);

A/sw/Bakum/8602/99 (H3N2); A/sw/Cotes d'Armor/604/99 (H1N2); A/sw/Cote

d'Armor/l 482/99 (HlNl); A/sw/Gent/7625/99 (H1N2); A/Hong Kong/1774/99 (H3N2);

A/sw/Hong Kong/5190/99 (H3N2); A/sw/Hong Kong/5200/99 (H3N2); A/sw/Hong

Kong/5212/99 (H3N2); A/sw/Ille et Villaine/1455/99 (HlNl); A/sw/Italy/1654- 1/99 (H1N2); A/sw/Italy/2034/99 (HlNl); A/sw/Italy/2064/99 (H1N2); A/sw/Berlin/1578/00 (H3N2); A sw/Bakum/1832/00 (H1N2); A sw/Bakum/1833/00 (H1N2); A/sw/Cote d'Armor/800/00 (H1N2); A/sw/Hong Kong/7982/00 (H3N2); A/sw/Italy/1081/00 (H1N2); A/sw/Belzig/2/01 (HlNl); A/sw/Belzig/54/01 (H3N2); A/sw/Hong Kong/9296/01 (H3N2); A/sw/Hong

Kong/9745/01 (H3N2); A/sw/Spain/33601/01 (H3N2); A/sw/Hong Kong/ 1144/02 (H3N2); A/sw/Hong Kong/1197/02 (H3N2); A/sw/Spain/39139/02 (H3N2); A/sw/Spain/42386/02 (H3N2); A/Switzerland/8808/2002 (HlNl); A/sw/Bakum/1769/03 (H3N2);

A/sw/Bissendorf/IDTl 864/03 (H3N2); A/sw/Ehren/IDT2570/03 (H1N2);

A/sw/Gescher/IDT2702/03 (H1N2); A/sw/Haselunne/2617/03 hp (HlNl);

A/sw/Loningen/IDT2530/03 (H1N2); A/sw/IVD/IDT2674/03 (H1N2);

A sw/Nordkirchen/IDT 1993/03 (H3N2); A/sw/Nordwalde/IDT2197/03 (H1N2);

A/sw/Norden/IDT2308/03 (H1N2); A/sw/Spain/50047/03 (HlNl); A/sw/Spain/51915/03 (HlNl); A/sw/Vechta/2623/03 (HlNl); A/sw/Visbek/IDT2869/03 (H1N2);

A/sw/Waltersdorf/IDT2527/03 (H1N2); A/sw/Damme/IDT2890/04 (H3N2);

A sw/Geldern/IDT2888/04 (HlNl); A/sw/Granstedt/IDT3475/04 (H1N2);

A/sw/Greven/IDT2889/04 (HlNl); A/sw/Gudensberg/IDT2930/04 (H1N2);

A/sw/Gudensberg/IDT2931/04 (H1N2); A/sw/Lohne/IDT3357/04 (H3N2);

A/sw/Nortrup/IDT3685/04 (H1N2); A/sw/Seesen/IDT3055/04 (H3N2); A/sw/Spain/53207/04 (HlNl); A/sw/Spain/54008/04 (H3N2); A/sw/Stolzenau/IDT3296/04 (H1N2);

A/sw/Wedel/IDT2965/04 (HlNl); A/sw/Bad Griesbach/IDT4191/05 (H3N2);

A/sw/Cloppenburg/IDT4777/05 (H1N2); A/sw/Dotlingen/IDT3780/05 (H1N2);

A/sw/Dotlingen/IDT4735/05 (H1N2); A/sw/Egglham/IDT5250/05 (H3N2);

A/sw/Harkenblek/IDT4097/05 (H3N2); A/sw/Hertzen/IDT4317/05 (H3N2);

A/sw/Krogel/IDT4192/05 (HlNl); A/sw/Laer/IDT3893/05 (HlNl); A/sw/Laer/IDT4126/05 (H3N2); A/sw/Merzen/IDT4114/05 (H3N2); A/sw/Muesleringen-S./IDT4263/05 (H3N2);

A/sw/Osterhofen/IDT4004/05 (H3N2); A/sw/Sprenge/IDT3805/05 (H1N2);

A/sw/Stadtlohn/IDT3853/05 (H1N2); A/sw/Voglarn/IDT4096/05 (HlNl);

A/sw/Wohlerst/IDT4093/05 (HlNl); A/sw/Bad Griesbach/IDT5604/06 (HlNl);

A/sw/Herzlake/IDT5335/06 (H3N2); A/sw/Herzlake/IDT5336/06 (H3N2);

A/sw/Herzlake/IDT5337/06 (H3N2); and A/wild b oar/Germany /R 169/2006 (H3N2).

[00345] Other specific examples of strains of influenza A virus include, but are not limited to: A/Toronto/3141/2009 (HlNl); A/Regensburg/D6/2009 (HlNl); A/Bayern/62/2009 (HlNl); A/Bayern/62/2009 (HlNl); A/Bradenburg/19/2009 (HlNl); A/Bradenburg/20/2009 (HlNl); A/Distrito Federal/2611/2009 (HlNl); A/Mato Grosso/2329/2009 (HlNl); A/Sao

Paulo/1454/2009 (HlNl); A/Sao Paulo/2233/2009 (HlNl); A/Stockholm/37/2009 (HlNl); A/Stockholm/41/2009 (HlNl); A/Stockholm/45/2009 (HlNl); A/swine/Alberta/OTH-33-1/2009 (HlNl); A/swine/Alberta/OTH-33-14/2009 (HlNl); A/swine/Alberta/OTH-33 -2/2009 (HlNl); A/swine/Alberta/OTH-33-21/2009 (HlNl); A/swine/Alberta/OTH-33 -22/2009 (HlNl);

A/swine/Alberta/OTH-33 -23/2009 (HlNl); A/swine/Alberta/OTH-33 -24/2009 (HlNl);

A swine/Alberta/OTH-33 -25/2009 (HlNl); A swine/Alberta/OTH-33 -3/2009 (HlNl);

A swine/Alberta/OTH-33 -7/2009 (HlNl); A Beijing/502/2009 (HlNl); A Firenze/ 10/2009 (HlNl); A/Hong Kong/2369/2009 (HlNl); A Italy/85/2009 (HlNl); A/Santo

Domingo/572N/2009 (HlNl); A Catalonia/385/2009 (HlNl); A Catalonia/386/2009 (HlNl); A Catalonia/387/2009 (HlNl); A Catalonia/390/2009 (HlNl); A Catalonia/394/2009

(HlNl);A Catalonia/397/2009 (HlNl); A Catalonia/398/2009 (HlNl); A Catalonia/399/2009 (HlNl); A/Sao Paulo/2303/2009 (HlNl); A Akita/ 1/2009 (HlNl); A Castro/JXP/2009 (HlNl); A Fukushima/1/2009 (HlNl); A Israel/276/2009 (HlNl); A Israel/277/2009 (HlNl);

A Israel/70/2009 (HlNl); A Iwate/ 1/2009 (HlNl); A/Iwate/2/2009 (HlNl);

A Kagoshima/1/2009 (HlNl); A Osaka/180/2009 (HlNl); A Puerto Montt/Bio87/2009 (HI Nl); A/Sao Paulo/2303/2009 (HlNl); A Sapporo/1/2009 (HlNl); A Stockholm/30/2009 (HlNl); A Stockholm/31/2009 (HlNl); A Stockholm/32/2009 (HlNl); A Stockholm/33/2009 (HlNl); A Stockholm/34/2009 (HlNl); A Stockholm/35/2009 (HlNl); A Stockholm/36/2009 (HlNl); A Stockholm/38/2009 (HlNl); A Stockholm/39/2009 (HlNl); A Stockholm/40/2009 (HlNl;) A Stockholm/42/2009 (HlNl); A Stockholm/43/2009 (HlNl); A Stockholm/44/2009 (HlNl); A Utsunomiya/2/2009 (HlNl); A WRAIR/0573N/2009 (HlNl); and

A Zhejiang/DTID-ZJU01/2009 (HlNl).

[00346] Non-limiting examples of influenza B viruses include strain Aichi/5/88, strain B/Brisbane/60/2008; Akita/27/2001, strain Akita/5/2001, strain Alaska/16/2000, strain

Alaska/1777/2005, strain Argentina/69/2001, strain Arizona/146/2005, strain Arizona/148/2005, strain Bangkok/163/90, strain Bangkok/34/99, strain Bangkok/460/03, strain Bangkok/54/99, strain Barcelona/215/03, strain Beijing/15/84, strain Beijing/184/93, strain Beijing/243/97, strain Beijing/43/75, strain Beijing/5/76, strain Beijing/76/98, strain Belgium/WVl 06/2002, strain Belgium/WVl 07/2002, strain Belgium/WVl 09/2002, strain Belgium/WVl 14/2002, strain Belgium/WV122/2002, strain Bonn/43, strain Brazil/952/2001, strain Bucharest/795/03, strain Buenos Aires/161/00), strain Buenos Aires/9/95, strain Buenos Aires/SW16/97, strain Buenos Aires/VL518/99, strain Canada/464/2001, strain Canada/464/2002, strain Chaco/366/00, strain Chaco/Rl 13/00, strain Cheju/303/03, strain Chiba/447/98, strain Chongqing/3/2000, strain clinical isolate SA1 Thailand/2002, strain clinical isolate SA10 Thailand/2002, strain clinical isolate SA100 Philippines/2002, strain clinical isolate SA101 Philippines/2002, strain clinical isolate SA110 Philippines/2002), strain clinical isolate SA112 Philippines/2002, strain clinical isolate SA113 Philippines/2002, strain clinical isolate SA114 Philippines/2002, strain clinical isolate SA2 Thailand/2002, strain clinical isolate SA20 Thailand/2002, strain clinical isolate SA38 Philippines/2002, strain clinical isolate SA39 Thailand/2002, strain clinical isolate SA99 Philippines/2002, strain CNIC/27/2001, strain Colorado/2597/2004, strain Cordoba/VA418/99, strain Czechoslovakia/16/89, strain Czechoslovakia/69/90, strain Daeku/10/97, strain

Daeku/45/97, strain Daeku/47/97, strain Daeku/9/97, strain B/Du/4/78, strain B/Durban/39/98, strain Durban/43/98, strain Durban/44/98, strain B/Durban/52/98, strain Durban/55/98, strain Durban/56/98, strain England/1716/2005, strain England/2054/2005) , strain England/23/04, strain Finland/154/2002, strain Finland/159/2002, strain Finland/160/2002, strain

Finland/161/2002, strain Finland/162/03, strain Finland/162/2002, strain Finland/162/91, strain Finland/164/2003, strain Finland/172/91, strain Finland/173/2003, strain Finland/176/2003, strain Finland/184/91, strain Finland/188/2003, strain Finland/190/2003, strain

Finland/220/2003, strain Finland/WV5/2002, strain Fujian/36/82, strain Geneva/5079/03, strain Genoa/11/02, strain Genoa/2/02, strain Genoa/21/02, strain Genova/54/02, strain Genova/55/02, strain Guangdong/05/94, strain Guangdong/08/93, strain Guangdong/5/94, strain

Guangdong/55/89, strain Guangdong/8/93, strain Guangzhou/7/97, strain Guangzhou/86/92, strain Guangzhou/87/92, strain Gyeonggi/592/2005, strain Hannover/2/90, strain Harbin/07/94, strain Hawaii/ 10/2001, strain Hawaii/ 1990/2004, strain Hawaii/38/2001, strain Hawaii/9/2001, strain Hebei/19/94, strain Hebei/3/94) , strain Henan/22/97, strain Hiroshima/23/2001, strain Hong Kong/ 110/99, strain Hong Kong/1115/2002, strain Hong Kong/112/2001, strain Hong Kong/123/2001, strain Hong Kong/1351/2002, strain Hong Kong/ 1434/2002, strain Hong Kong/ 147/99, strain Hong Kong/ 156/99, strain Hong Kong/ 157/99, strain Hong Kong/22/2001, strain Hong Kong/22/89, strain Hong Kong/336/2001, strain Hong Kong/666/2001, strain Hong Kong/9/89, strain Houston/1/91, strain Houston/1/96, strain Houston/2/96, strain Hunan/4/72, strain Ibaraki/2/85, strain ncheon/297/2005, strain India/3/89, strain India/77276/2001, strain Israel/95/03, strain Israel/WV 187/2002, strain Japan/1224/2005, strain Jiangsu/ 10/03, strain Johannesburg/1/99, strain Johannesburg/96/01, strain Kadoma/1076/99, strain Kadoma/122/99, strain Kagoshima/ 15/94, strain Kansas/22992/99, strain Khazkov/224/91, strain Kobe/1/2002, strain, strain Kouchi/193/99, strain Lazio/1/02, strain Lee/40, strain Leningrad/129/91, strain Lissabon/2/90) , strain Los Angeles/1/02, strain Lusaka/270/99, strain Lyon/1271/96, strain Malaysia/83077/2001, strain Maputo/ 1/99, strain Mar del Plata/595/99, strain Maryland/ 1/01, strain Memphis/ 1/01, strain Memphis/ 12/97-MA, strain Michigan/22572/99, strain Mie/1/93, strain Milano/1/01, strain Minsk/318/90, strain Moscow/3/03, strain Nagoya/20/99, strain Nanchang/1/00, strain Nashville/107/93, strain Nashville/45/91, strain Nebraska/2/01, strain Netherland/801/90, strain Netherlands/429/98, strain New York/ 1/2002, strain NIB/48/90, strain Ningxia/45/83, strain Norway/1/84, strain Oman/ 16299/2001, strain Osaka/1059/97, strain Osaka/983/97-V2, strain Oslo/1329/2002, strain Oslo/1846/2002, strain Panama/45/90, strain Paris/329/90, strain Parma/23/02, strain Perth/211/2001, strain Peru/ 1364/2004, strain

Philippines/5072/2001, strain Pusan/270/99, strain Quebec/173/98, strain Quebec/465/98, strain Quebec/7/01, strain Roma/1/03, strain Saga/S172/99, strain Seoul/13/95, strain Seoul/37/91, strain Shangdong/7/97, strain Shanghai/361/2002) , strain Shiga/T30/98, strain Sichuan/379/99, strain Singapore/222/79, strain Spain/WV27/2002, strain Stockholm/ 10/90, strain

Switzerland/5441/90, strain Taiwan/0409/00, strain Taiwan/0722/02, strain Taiwan/97271/2001, strain Tehran/80/02, strain Tokyo/6/98, strain Trieste/28/02, strain Ulan Ude/4/02, strain United Kingdom/34304/99, strain USSR/100/83, strain Victoria/103/89, strain Vienna/1/99, strain Wuhan/356/2000, strain WV194/2002, strain Xuanwu/23/82, strain Yamagata/1311/2003, strain Yamagata/K500/2001, strain Alaska/12/96, strain GA/86, strain NAGASAKI/1/87, strain Tokyo/942/96, strain B/Wisconsin/1/2010; and strain Rochester/02/2001. In a specific embodiment, the influenza B virus is B/Malaysia/2506/04.

[00347] Other examples of influenza viruses may be found elsewhere in the application, such as in, e.g., Section 5.1 above and Section 6 below.

[00348] In certain embodiments, the influenza viruses provided herein have an attenuated phenotype. In specific embodiments, the attenuated influenza virus is based on influenza A virus. In other embodiments, the attenuated influenza virus is based on influenza B virus. In yet other embodiments, the attenuated influenza virus is based on influenza C virus. In other embodiments, the attenuated influenza virus may comprise genes or genome segments from one or more strains or subtypes/lineages of influenza A, influenza B, and/or influenza C virus. In some embodiments, the attenuated backbone virus comprises genes from an influenza A virus and an influenza B virus. In specific embodiments, the attenuated influenza virus comprises, encodes, or both, a chimeric HA and has a backbone of an influenza A virus. In specific embodiments, the attenuated influenza virus comprises, encodes, or both, a chimeric HA and has a backbone of an influenza B virus.

[00349] In specific embodiments, attenuation of influenza virus is desired such that the virus remains, at least partially, infectious and can replicate in vivo, but only generate low titers resulting in subclinical levels of infection that are non-pathogenic. Such attenuated viruses are especially suited for embodiments described herein wherein the virus or an immunogenic composition thereof is administered to a subject to induce an immune response. Attenuation of the influenza virus can be accomplished according to any method known in the art, such as, e.g., selecting viral mutants generated by chemical mutagenesis, mutation of the genome by genetic engineering, selecting reassortant viruses that contain segments with attenuated function {e.g., truncated NS1 protein (see, e.g., Hai et al, 2008, Journal of Virology 82(21): 10580-10590, which is incorporated by reference herein in its entirety) or NS1 deletion (see, e.g., Wressnigg et al, 2009, Vaccine 27:2851-2857, which is incorporated by reference herein in its entirety)), or selecting for conditional virus mutants (e.g., cold-adapted viruses, see, e.g., Alexandrova et al, 1990, Vaccine, 8:61-64, which is incorporated by reference herein in its entirety). Alternatively, naturally occurring attenuated influenza viruses may be used as influenza virus backbones for the influenza virus vectors.

[00350] In certain embodiments, an influenza virus comprising a chimeric HA described herein has one, two, or more of the functions of an influenza virus comprising a wild-type influenza virus HA. Nonlimiting examples of functions of a wild-type influenza virus HA include fusogenic activity, receptor binding activity, budding, and particle formation. In a specific embodiment, an influenza virus comprising a chimeric influenza HA polypeptide described herein has fusogenic activity. Assays known to one skilled in the art can be utilized to assess the fusogenic activity of an influenza virus comprising a chimeric influenza HA polypeptide described herein, such as, for example, immunofluorescence assays and pseudotyped virus-like-particle assays. In a specific embodiment, an influenza virus comprising a chimeric influenza HA polypeptide described herein has replication activity. Assays known to one skilled in the art can be utilized the assess the replication activity of an influenza virus comprising a chimeric influenza HA polypeptide described herein, such as, for example, plaque assay and western blot analyses.

5.7 VIRUS-LIKE PARTICLES AND VIROSOMES

[00351] The chimeric influenza virus hemagglutinin polypeptides described herein can be incorporated into virus-like particle (VLP) vectors, e.g., purified/isolated VLPs. VLPs generally comprise a viral polypeptide(s) typically derived from a structural protein(s) of a virus. In some embodiments, the VLPs are not capable of replicating. In certain embodiments, the VLPs may lack the complete genome of a virus or comprise a portion of the genome of a virus. In some embodiments, the VLPs are not capable of infecting a cell. In some embodiments, the VLPs express on their surface one or more of viral (e.g., virus surface glycoprotein) or non-viral (e.g., antibody or protein) targeting moieties known to one skilled in the art or described herein. In some embodiments, the VLPs comprise a chimeric hemagglutinin (HA) polypeptide and a viral structural protein, such as HIV gag. In a specific embodiment, the VLPs comprise a chimeric hemagglutinin (HA) polypeptide and an HIV gag polypeptide. In another specific embodiment, the VLPs comprise a chimeric hemagglutinin (HA) polypeptide and influenza virus

neuraminidase polypeptide. In another specific embodiment, the VLPs comprise a chimeric hemagglutinin (HA) polypeptide, influenza virus neuraminidase polypeptide, and influenza virus Ml polypeptide.

[00352] Methods for producing and characterizing recombinantly produced VLPs have been described based on several viruses, including influenza virus (Bright et al. (2007) Vaccine. 25:3871), human papilloma virus type 1 (Hagnesee et al. (1991) J. Virol. 67:315), human papilloma virus type 16 (Kirnbauer et al. Proc. Natl. Acad. Sci. (1992)89: 12180), HIV-1 (Haffer et al, (1990) J. Virol. 64:2653), and hepatitis A (Winokur (1991) 65:5029), each of which is incorporated herein in its entirety. Methods for expressing VLPs that contain NDV proteins are provided by Pantua et al. (2006) J. Virol. 80: 11062-11073, and in United States patent application Publication No. 20090068221, published March 12, 2009, each of which is incorporated in its entirety herein. In a specific embodiment, the VLPs comprising chimeric hemagglutinin (HA) polypeptide described herein are generated using baculovirus, as described in the Examples section below. In other embodiments, the VLPs comprising chimeric hemagglutinin (HA) polypeptides described herein are generated using 293T cells.

[00353] In specific embodiments, VLPs, e.g., VLPs comprising a chimeric hemagglutinin (HA) polypeptide are expressed in cells (such as, e.g., mammalian cells (e.g., 293T cells) and insect cells (e.g., High Five cells and Sf9 cells). In certain embodiments, the VLPs are expressed in cells that express surface glycoproteins that comprise sialic acid. In certain embodiments, VLPs, e.g., VLPs comprising a chimeric hemagglutinin (HA) polypeptide, are expressed in cells that do not express surface glycoproteins that comprise sialic acid.

[00354] In a specific embodiment, a chimeric hemagglutinin (HA) polypeptide may be incorporated into a virosome. A virosome containing a chimeric hemagglutinin (HA) polypeptide may be produced using techniques known to those skilled in the art. For example, a virosome may be produced by disrupting a purified virus, extracting the genome, and

reassembling particles with the viral proteins (e.g., a chimeric hemagglutinin (HA) polypeptide) and lipids to form lipid particles containing viral proteins.

[00355] Also provided herein are methods for producing and characterizing recombinantly produced VLPs comprising a chimeric HA described herein. Methods for producing and characterizing recombinantly produced VLPs have been described based on several viruses, including influenza virus (Bright et al. (2007) Vaccine. 25:3871), human papilloma virus type 1 (Hagnesee et al. (1991) J. Virol. 67:315), human papilloma virus type 16 (Kirnbauer et al. Proc. Natl. Acad. Sci. (1992)89: 12180), HIV- 1 (Haffer et al, (1990) J. Virol. 64:2653), and hepatitis A (Winokur (1991) 65:5029), each of which is incorporated herein in its entirety. In a specific embodiment, the VLPs comprising chimeric HA polypeptides described herein are generated using baculovirus. In other embodiments, the VLPs comprising chimeric HA polypeptides described herein are generated using 293T cells.

[00356] In specific embodiments, VLPs, e.g., VLPs comprising a chimeric HA polypeptide, are expressed in cells (e.g., 293T cells). In certain embodiments, the VLPs are expressed in cells that express surface glycoproteins that comprise sialic acid. In accordance with such

embodiments, the cells are cultured in the presence of neuraminidase (e.g., viral of bacterial neuraminidase). In certain embodiments, VLPs, e.g., VLPs comprising a chimeric HA polypeptide, are expressed in cells that do not express surface glycoproteins that comprise sialic acid. [00357] In a specific embodiment, a chimeric HA polypeptide may be incorporated into a virosome. A virosome containing a chimeric HA polypeptide may be produced using techniques known to those skilled in the art. For example, a virosome may be produced by disrupting a purified virus, extracting the genome, and reassembling particles with the viral proteins (e.g., a chimeric HA polypeptide) and lipids to form lipid particles containing viral proteins.

5.8 COMPOSITIONS

[00358] The nucleic acids, vectors, and polypeptides described herein (sometimes referred to herein as "active compounds") may be incorporated into compositions. In specific embodiments, an active compound described herein is a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s). In a specific embodiment, the

compositions are pharmaceutical compositions, such as immunogenic compositions (e.g., vaccine formulations). The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject. In a specific embodiment, the pharmaceutical compositions are suitable for veterinary and/or human administration. The compositions may be used in methods of preventing or treating an influenza virus disease. The compositions may be used in methods to induce an immune response against influenza virus. The compositions may be used in methods to immunize against influenza virus.

[00359] In a specific embodiment, a pharmaceutical composition (e.g., immunogenic composition) comprises a chimeric influenza virus hemagglutinin polypeptide in an admixture with a pharmaceutically acceptable carrier. In a specific embodiment, a pharmaceutical composition (e.g., immunogenic composition) comprises a chimeric influenza virus

hemagglutinin polypeptide and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below), in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide described herein, in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a

pharmaceutical composition (e.g., an immunogenic composition) comprises a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide described herein and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below), in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises an expression vector comprising a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below), in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises an expression vector comprising a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide, in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises an influenza virus or non-influenza virus containing a chimeric hemagglutinin (HA) polypeptide, in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below) and an influenza virus or non-influenza virus containing a chimeric hemagglutinin (HA) polypeptide, in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises an influenza virus or non-influenza virus having a genome engineered to express a chimeric hemagglutinin (HA) polypeptide, in admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below) and an influenza virus or non- influenza virus having a genome engineered to express a chimeric hemagglutinin (HA) polypeptide, in admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises a virus-like particle or virosome containing a chimeric hemagglutinin (HA) polypeptide, in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises a virus-like particle or virosome containing a chimeric hemagglutinin (HA) polypeptide and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below), in an admixture with a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises a bacteria expressing or engineered to express a chimeric hemagglutinin (HA) polypeptide, in an admixture with a pharmaceutically acceptable carrier. In another

embodiment, a pharmaceutical composition (e.g., an immunogenic composition) comprises a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below) and a bacteria expressing or engineered to express a chimeric hemagglutinin (HA) polypeptide, in an admixture with a pharmaceutically acceptable carrier. In a specific embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 below and Section 6 below)

[00360] In some embodiments, a pharmaceutical composition may comprise one or more other therapies in addition to a therapy that utilizes a chimeric hemagglutinin (HA) polypeptide described. In certain embodiments, a chimeric HA polypeptide, nucleic acid sequence encoding a chimeric HA polypeptide, or a vector containing, expressing, or both such a polypeptide(s) is the sole active ingredient in a pharmaceutical composition (e.g., an immunogenic composition).

[00361] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the

pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should suit the mode of administration.

[00362] In a specific embodiment, pharmaceutical compositions are formulated to be suitable for the intended route of administration to a subject. For example, the pharmaceutical composition may be formulated to be suitable for parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration. In a specific embodiment, the

pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration. In a specific embodiment, the pharmaceutical composition may be formulated for intramuscular administration. In a specific embodiment, the pharmaceutical composition may be formulated for subcutaneous administration. In a specific embodiment, the

pharmaceutical composition may be formulated for intranasal administration. [00363] In specific embodiments, immunogenic compositions described herein are monovalent formulations. In other embodiments, immunogenic compositions described herein are multivalent formulations. In one example, a multivalent formulation comprises more than one vector expressing a chimeric hemagglutinin (HA) polypeptide. In another example, a multivalent formulation comprises more than one virus containing a chimeric hemagglutinin (HA) polypeptide. In certain embodiments, a multivalent formulation may comprise one, two, three or more different chimeric hemagglutinin (HA) polypeptides expressed using a single vector. In certain embodiments, immunogenic compositions described herein are trivalent vaccines which comprise at least one chimeric hemagglutinin (HA) polypeptide. In some embodiments, immunogenic compositions described herein are trivalent vaccines which comprise three different influenza viruses, each influenza virus comprising a different chimeric HA. In some embodiments, immunogenic compositions described herein are quadrivalent vaccines which comprise at least two different chimeric hemagglutinin (HA) polypeptides, described herein. In some embodiments, immunogenic compositions described herein are quadrivalent vaccines which comprise four different influenza viruses, each influenza virus comprising a different chimeric HA. In certain embodiments, a composition described herein comprises one or more of the chimeric hemagglutinin (HA) polypeptides described in

International Patent Application Publication Nos. WO 2014/099931, WO 2013/043729 and WO 2017/218624, U.S. Patent Nos. 9,371,366 and 9,968,670, U.S. Patent Application Publication No. 2015/0132330 and 2018/0008696 (each of which is incorporated herein by reference in its entirety), and one or more of the chimeric HA polypeptides described herein. In certain embodiments, a composition described herein comprises 2, 3, or more of the chimeric HA polypeptides described herein. In certain embodiments, a compositions described herein may comprise a chimeric HA described herein in Section 5.1.1 (e.g., a chimeric influenza virus HA polypeptide described in one, two, or all of the Sections 5.1.1.1, 5.1.1.2, or 5.1.1.3 above), and a chimeric HA described in Section Error! Reference source not found.. In specific

embodiments, an immunogenic composition described herein comprises a first chimeric HA polypeptide, a second chimeric HA polypeptide and a third chimeric HA polypeptide, wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or HI 6), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, HI 4, or HI 5), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises the a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of an influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the influenza B virus HA globular head domain, and the third HA stem domain comprises the HA stem domain of the influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain. In specific embodiments, an immunogenic composition described herein comprises a first chimeric HA polypeptide, a second chimeric HA polypeptide, a third chimeric HA polypeptide and adjuvant (in specific embodiments, a liposomal adjuvant such as described in Section 5.8.5 below or Section 6 below), wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H5, H7, H10, H14, or H15), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises the a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of an influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the influenza B virus HA globular head domain, and the third HA stem domain comprises the HA stem domain of the influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain. The immunogenic composition may be an inactivated vaccine, such as subunit vaccine, split vaccine or whole inactivated virus vaccine. In particular embodiments, the first and second chimeric HA polypeptides may be ones described in Section 5.1 above (in particular, Section 5.1.1.1 above), Section 6.1 below, Section 6.2 below, Section 6.3 below, or Section 6.4 below and the third chimeric HA polypeptide may be one described in Section 5.1.2 above or Section 6.5 below. In a specific embodiment, the second HA subtype is HI and the fourth HA subtype is H3. In another specific embodiment, the influenza B virus is B/Yamagata/16/88. In a specific embodiment, the amino acid substitutions in the one, two, three or all of the loops and helix of the globular head domain of the influenza virus B virus HA correspond to to amino acid residues found in a corresponding regionof the globular head domain of an influenza A virus (e.g., an influena A virus of an H5, H8, HI 1 or H13 subtype).

[00364] An immunogenic composition described herein may be used to immunize a subject against influenza virus. An immunogenic composition described herein may also be used to prevent, treat or both an influenza virus infection or an influenza virus disease in a subject.

[00365] In certain embodiments, the pharmaceutical compositions (e.g., immunogenic compositions) described herein additionally comprise one or more components used to inactivate a virus, e.g., formalin or formaldehyde or a detergent such as sodium deoxycholate, octoxynol 9 (Triton X-100), and octoxynol 10. In other embodiments, the pharmaceutical compositions described herein do not comprise any components used to inactivate a virus.

[00366] In certain embodiments, the pharmaceutical compositions (e.g., immunogenic compositions) described herein additionally comprise one or more buffers, e.g., phosphate buffer and sucrose phosphate glutamate buffer. In other embodiments, the pharmaceutical

compositions described herein do not comprise buffers.

[00367] The pharmaceutical compositions (e.g., immunogenic compositions) described herein can be included in a container, pack, or dispenser together with instructions for administration.

[00368] The pharmaceutical compositions (e.g., immunogenic compositions) described herein can be stored before use, e.g., the pharmaceutical compositions can be stored frozen (e.g., at about -20°C or at about -70°C); stored in refrigerated conditions (e.g., at about 4°C); or stored at room temperature (see International Application No. PCT/IB2007/001 149 published as

International Publication No. WO 07/1 10776, which is herein incorporated by reference in its entirety, for methods of storing compositions comprising influenza vaccines without refrigeration).

[00369] In a specific embodiment, an immunogenic composition is an inactivated vaccine comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below) and a chimeric HA polypeptide. The inactivated vaccine may be a whole virus inactivated vaccine, split virion vaccine, or subunit vaccine. Techniques for producing such vaccines are known to one of skill in the art.

[00370] In a specific embodiment, an immunogenic composition (e.g., a vaccine formulation) is an inactivated influenza virus that comprises a chimeric HA polypeptide and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below). In a specific

embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 below and Section 6 below).

[00371] In a specific embodiment, an immunogenic composition (e.g., a vaccine formulation) is one described in Section 6 below.

[00372] In specific embodiments, a vaccine formulation is a subunit vaccine that comprises a chimeric HA polypeptide and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below). In specific embodiments, a vaccine formulation is a split vaccine that comprises a chimeric HA polypeptide and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below). In certain embodiments, a vaccine formulation is a VLP that comprises a chimeric HA polypeptide and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below). In a specific embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 below and Section 6 below).

[00373] In certain embodiments, an immunogenic composition (e.g., a vaccine formulation) is multivalent. In one embodiment, an immunogenic composition (e.g., a vaccine formulation) comprises three chimeric HAs and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below), wherein the first chimeric HA comprises a stem domain polypeptide from an HI influenza virus and a first HA globular head domain, the second chimeric HA comprises a stem domain polypeptide from an H3 influenza virus and a second HA globular head domain, and the third chimeric HA comprises a stem domain polypeptide from an influenza B virus and a third HA globular head domain, wherein the first, second and third HA globular head domains are each from a different subtype or strain of influenza virus hemagglutinin, and wherein the HA globular head domain of each chimeric HA is heterologous to the stem domain polypeptide of each chimeric HA. In another specific embodiment, the first HA globular head domain is from an influenza A virus H5 subtype, H8 subtype, HI 1 subtype, HI 2 subtype, or another influenza A virus subtype (see, e.g., Section 5.1.1, supra and Section 6, infra). In another specific embodiment, the second HA globular head domain is from an influenza A virus H4 subtype, H5 subtype, H7 subtype, H14 subtype, HI 5 subtype, or another influenza A virus subtype (see, e.g., Section 5.1.1, supra and Section 6, infra). In another specific embodiment, the third chimeric HA is one described in Section 5.1.2, supra. In a specific embodiment, the liposomal adjuvant is a liposomal adjuvant {e.g., a liposomal adjuvant described in Section 5.8.5 below) {see, e.g., Section 5.8.5 below and Section 6 below).

[00374] In one embodiment, an immunogenic composition {e.g., a vaccine formulation) comprises three vectors and a liposomal adjuvant {e.g., a liposomal adjuvant described in Section 5.8.5 below), wherein each vector comprises a chimeric HA, wherein the first vector comprises a first chimeric HA comprising a stem domain polypeptide from an HI influenza virus and a first HA globular head domain, the second vector comprises a second chimeric HA comprising a stem domain polypeptide from an H3 influenza virus and a second HA globular head domain, and the third vector comprises a third chimeric HA comprising a stem domain polypeptide from an influenza B virus and a third HA globular head domain, wherein the first, second and third HA globular head domains are each from a different subtype or strain of influenza virus

hemagglutinin, and wherein the HA globular head domain of each chimeric HA is heterologous to the stem domain polypeptide of each chimeric HA. In a specific embodiment, the liposomal adjuvant is AS01 {see, e.g., Section 5.8.5 below and Section 6 below). In another specific embodiment, the first HA globular head domain is from an influenza A virus H5 subtype, H8 subtype, HI 1 subtype, H12 subtype, or another influenza A virus subtype (see, e.g., Section 5.1.1, supra and Section 6, infra). In another specific embodiment, the second HA globular head domain is from an influenza A virus H4 subtype, H5 subtype, H7 subtype, H14 subtype, HI 5 subtype, or another influenza A virus subtype (see, e.g., Section 5.1.1, supra and Section 6, infra). In another specific embodiment, the third chimeric HA is one described in Section 5.1.2, supra. In certain embodiments, the vector is a viral vector (e.g., an influenza virus) or VLP. See, e.g., Sections 5.6 above for examples of influenza virus vectors. In some embodiments, the viral vector is inactivated. [00375] In a specific embodiment, an immunogenic composition (e.g., a vaccine formulation) described herein comprises a live attenuated influenza virus engineered to express a chimeric hemagglutinin (HA), wherein the chimeric HA comprises an 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 for use in a prophylactic or therapeutic vaccine against influenza infection. In other embodiments, an immunogenic composition (e.g., a vaccine formulation) described herein comprises an inactivated influenza virus and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below), wherein the inactivated influenza virus comprises a chimeric HA, wherein the chimeric HA comprises a second influenza virus HA globular head domain and the HA stem domain, wherein the globular head domain is heterologous to the HA stem domain, for use in a prophylactic or therapeutic vaccine against influenza infection. In a specific embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 below and Section 6 below). In a specific embodiment, the immunogenic composition (e.g., a vaccine formulation)is for use in a prophylactic or therapeutic vaccine comprises an influenza virus hemagglutinin globular head domain from an influenza A virus of subtype H5, H8, HI 1 or H12 virus and a stem domain from a HI virus. In another embodiment, the immunogenic composition (e.g., a vaccine formulation) is for use in a prophylactic or therapeutic vaccine comprises an influenza virus hemagglutinin globular head domain globular head domain from an influenza A virus of subtype H4, H7, HI 4 or HI 5 virus and a stem domain from a H3 virus.

[00376] In a specific embodiment, an immunogenic composition described herein induces influenza virus-specific T cells in the lung of an animal as assessed by techniques known to one of skill in the art or described herein. See, e.g., Section 6, infra. In another specific embodiment, an immunogenic composition described herein is one described in Section 6, infra.

5.8.1 Subunit Vaccines

[00377] In a specific embodiment, provided herein are subunit vaccines comprising a chimeric influenza virus hemagglutinin polypeptide described herein. In a specific embodiment, provided herein are subunit vaccines comprising a chimeric influenza virus hemagglutinin polypeptide described herein and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below). In some embodiments, a subunit vaccine comprises a chimeric hemagglutinin (HA) polypeptide, one or more surface glycoproteins (e.g., influenza virus neuraminidase), other targeting moieties, and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below). In a specific embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 below and Section 6 below). In specific embodiments, a subunit vaccine comprises a single chimeric hemagglutinin (HA) polypeptide. In other embodiments, a subunit vaccine comprises two, three, four or more chimeric hemagglutinin (HA) polypeptides. In specific embodiments, the chimeric hemagglutinin (HA) polypeptide(s) used in a subunit vaccine are not membrane- bound, i.e., are soluble. In specific embodiments, the polypeptide components of the subunit vaccine are generated in a baculovirus expression system. In a particular embodiment, a subunit vaccine comprises a purified chimeric HA polypeptide described herein which is produced in a continuous insect cell line, such as one derived from the fall armyworm Spodoptera frugiperda using a baculovirus vector (e.g., Autographa californica nuclear polyhedrosis virus). The chimeric HA polypeptide may be extracted from the cells and further purified by column chromatography. In some embodiments, a subunit vaccine comprises more than one chimeric HA polypeptide described herein.

[00378] In a specific embodiment, the subunit vaccine is prepared using influenza virus that was propagated in embryonated chicken eggs (i.e., the components of the subunit vaccine (e.g., a chimeric hemagglutinin (HA) polypeptide) are isolated from virus that was propagated in embryonated chicken eggs). In another specific embodiment, the subunit vaccine is prepared using influenza virus that was not propagated in embryonated chicken eggs (i.e., the components of the subunit vaccine (e.g., a chimeric hemagglutinin (HA) polypeptide) are isolated from virus that was not propagated in embryonated chicken eggs). In another specific embodiment, the subunit vaccine is prepared using influenza virus that was propagated in mammalian cells, e.g., immortalized human cells (see, e.g., International Application No. PCT/EP2006/067566 published as International Publication No. WO 07/045674 which is herein incorporated by reference in its entirety) or canine kidney cells such as MDCK cells (see, e.g., International Application No. PCT/IB2007/003536 published as International Publication No. WO 08/032219 which is herein incorporated by reference in its entirety) (i.e., the components of the subunit vaccine (e.g., a chimeric hemagglutinin (HA) polypeptide) are isolated from virus that was propagated in mammalian cells). In another specific embodiment, the chimeric hemagglutinin (HA) polypeptide(s) in a subunit vaccine are prepared using an expression vector, e.g., a viral vector, plant vector, or baculovirus vector (i.e., the chimeric hemagglutinin (HA) polypeptide(s) in the subunit vaccine are obtained/isolated from an expression vector).

5.8.2 Live Virus Vaccines

[00379] In one embodiment, provided herein are immunogenic compositions (e.g., vaccines) comprising a live influenza virus containing a chimeric influenza virus hemagglutinin polypeptide. In another embodiment, provided herein are immunogenic compositions (e.g., vaccines) comprising a live virus that is engineered to encode a chimeric hemagglutinin (HA) polypeptide, which is expressed by progeny virus produced in the subjects administered the compositions. In certain embodiments, such immunogenic compositions may further comprise a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below). In specific embodiments, the chimeric hemagglutinin (HA) polypeptide is membrane-bound. In other specific embodiments, the chimeric hemagglutinin (HA) polypeptide is not membrane-bound, i.e., it is soluble. In particular embodiments, the live virus is an influenza virus, such as described in Section 5.6 above. In some embodiments, the live virus is attenuated. In some embodiments, an immunogenic composition comprises two, three, four or more live viruses containing or engineered to express two, three, four or more different chimeric hemagglutinin (HA) polypeptides.

[00380] An immunogenic composition comprising a live influenza virus for administration to a subject may be preferred because multiplication of the virus in the subject may lead to a prolonged stimulus of similar kind and magnitude to that occurring in natural infections, and therefore, confer substantial, long lasting immunity.

[00381] In a specific embodiment, the live virus that contains a chimeric hemagglutinin (HA) polypeptide is propagated in embryonated chicken eggs before its use in an immunogenic composition described herein. In another specific embodiment, the live virus that contains a chimeric hemagglutinin (HA) polypeptide is not propagated in embryonated chicken eggs before its use in an immunogenic composition described herein. In another specific embodiment, the live virus that contains a chimeric hemagglutinin (HA) polypeptide is propagated in mammalian cells, e.g., immortalized human cells (see, e.g., International Application No.

PCT/EP2006/067566 published as International Publication No. WO 07/045674 which is herein incorporated by reference in its entirety) or canine kidney cells such as MDCK cells (see, e.g., International Application No. PCT/IB2007/003536 published as International Publication No. WO 08/032219 which is herein incorporated by reference in its entirety) before its use in an immunogenic composition described herein.

5.8.3 Inactivated Virus Vaccines

[00382] In one embodiment, provided herein are immunogenic compositions (e.g., vaccines) comprising an inactivated virus containing a chimeric influenza virus hemagglutinin polypeptide. In one embodiment, provided herein are immunogenic compositions (e.g., vaccines) comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 below) and an inactivated virus containing a chimeric influenza virus hemagglutinin polypeptide. In a specific embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 below and Section 6 below). In specific embodiments, the chimeric hemagglutinin (HA) polypeptide is membrane- bound. In particular embodiments, the inactivated virus is an influenza virus, such as described in Section 5.6 above. In some embodiments, an immunogenic composition comprises two, three, four or more inactivated viruses containing two, three, four or more different chimeric hemagglutinin (HA) polypeptides. In certain embodiments, the inactivated virus immunogenic compositions comprise one or more adjuvants.

[00383] Techniques known to one of skill in the art may be used to inactivate viruses containing a chimeric hemagglutinin (HA) polypeptide. Common methods use formalin, heat, or detergent for inactivation. See, e.g., U.S. Patent No. 6,635,246, which is herein incorporated by reference in its entirety. Other methods include those described in U.S. Patent Nos. 5,891,705; 5, 106,619, 4,693,981, 7,238,349, and 7,316,813, U.S. Patent Application Publication Nos.

2008/0181911 and 2009/0263422, and International Patent Application Publication Nos. WO 2001/022992, WO 2006/100109, WO 2002/097072, and WO 2008/009309, each which are incorporated herein by reference in their entireties.

[00384] In a specific embodiment, the inactivated virus that contains a chimeric

hemagglutinin (HA) polypeptide was propagated in embryonated chicken eggs before its inactivation and subsequent use in an immunogenic composition described herein. In another specific embodiment, the inactivated virus that contains a chimeric hemagglutinin (HA) polypeptide was not propagated in embryonated chicken eggs before its inactivation and subsequent use in an immunogenic composition described herein. In another specific

embodiment, the inactivated virus that contains a chimeric hemagglutinin (HA) polypeptide was propagated in mammalian cells, e.g., immortalized human cells (see, e.g., International

Application No. PCT/EP2006/067566 published as International Publication No. WO

07/045674 which is herein incorporated by reference in its entirety) or canine kidney cells such as MDCK cells (see, e.g., International Application No. PCT/IB2007/003536 published as International Publication No. WO 08/032219 which is herein incorporated by reference in its entirety) before its inactivation and subsequent use in an immunogenic composition described herein.

5.8.4 Split Virus Vaccines

[00385] In one embodiment, an immunogenic composition comprising a chimeric influenza virus hemagglutinin polypeptide is a split virus vaccine. In one embodiment, an immunogenic composition comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section

5.8.5 below) and a chimeric influenza virus hemagglutinin polypeptide is a split virus vaccine. In a specific embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 below and Section 6 below). In some embodiments, split virus vaccine contains two, three, four or more different chimeric hemagglutinin (HA) polypeptides. In certain embodiments, the chimeric hemagglutinin (HA) polypeptide and/or the influenza virus neuraminidase polypeptide is/was membrane-bound. In specific embodiments, the split virus vaccine is one or more of the split virus vaccines descried in Section 6 below.

[00386] Techniques for producing split virus vaccines are known to those skilled in the art. By way of non-limiting example, an influenza virus split vaccine may be prepared using inactivated particles disrupted with detergents. One example of a split virus vaccine that can be adapted for use in accordance with the methods described herein is the fluzone®, Influenza Virus Vaccine (Zonal Purified, Subvirion) for intramuscular use, which is formulated as a sterile suspension prepared from influenza viruses propagated in embryonated chicken eggs. The virus- containing fluids are harvested and inactivated with formaldehyde. Influenza virus is

concentrated and purified in a linear sucrose density gradient solution using a continuous flow centrifuge. The virus is then chemically disrupted using a nonionic surfactant, octoxinol-9, (Triton® X- 100 - A registered trademark of Union Carbide, Co.) producing a "split virus." The split virus is then further purified by chemical means and suspended in sodium phosphate- buffered isotonic sodium chloride solution. [00387] In a specific embodiment, the split virus vaccine is prepared using influenza virus that was propagated in embryonated chicken eggs. In another specific embodiment, the split virus vaccine is prepared using influenza virus that was not propagated in embryonated chicken eggs. In another specific embodiment, the split virus vaccine is prepared using influenza virus that was propagated in mammalian cells, e.g., immortalized human cells (see, e.g., PCT/EP2006/067566 published as WO 07/045674 which is herein incorporated by reference in its entirety) or canine kidney cells such as MDCK cells (see, e.g., PCT/IB2007/003536 published as WO 08/032219 which is herein incorporated by reference in its entirety). Other methods for preparing the split virus vaccine are known in the art, such as, e.g., those described in U.S. Patent Nos. 7,238,349 and 7,316,813, U.S. Patent Application Publication Nos. 2008/0181911 and 2009/0263422, and International Patent Application Publication Nos. WO 2001/022992, WO 2006/100109, WO 2002/097072, and WO 2008/009309, each which are incorporated herein by reference in their entireties.

5.8.5 Liposomal Adjuvants

[00388] In preferred embodiments, the compositions described herein comprise, or are administered in combination with, a liposomal adjuvant (see, e.g., AS01 in Section 6 below). In certain embodiments, the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. In certain embodiments, the liposomal adjuvant comprises a TLR4 agonist and saponin. In certain embodiments, the TLR4 agonist is a lipopolysaccharide. In certain embodiments, the TLR4 agonist is a non-toxic derivative of lipid A. In certain embodiments, the TLR4 agonist is a monophosphoryl lipid A. In certain embodiments, the TLR4 agonist is 3-de-O-acylated monophosphoryl lipid A ("MPL"). In certain embodiments, the saponin is obtainable from Quillaja saponaria. In certain embodiments, the saponin comprises one or more of QS7, QS17, QS18 and QS21. In certain embodiments, the saponin comprises QS21. In certain embodiments, the TLR4 agonist is 3-O-desacyl-monophosphoiyl lipid A and the saponin is QS21.

[00389] In certain embodiments, the liposomes comprise l,2-dioleoyl-sn-glycero-3- phosphatidylcholine ("DOPC"). In certain embodiments, the liposomes comprise cholesterol. In certain embodiments, the liposomes comprise DOPC and cholesterol.

[00390] In certain embodiments, the liposomal adjuvant is diluted in a pharmaceutically acceptable excipient. In certain embodiments, the adjuvant is diluted in a pharmaceutically acceptable excipient, such that a composition comprising the diluted liposomal adjuvant and a chimeric influenza virus HA polypeptide described herein, or a composition therof, has a suitable pH and osmolarity for the intended route of administration of the composition. In certain embodiments, the liposomal adjuvant is diluted in phosphate buffered saline. In certain embodiments, the liposomal adjuvant is diluted in sorbitol. In certain embodiments, the pH of the diluted liposomal adjuvant is between 6 and 7, 6.1 and 6.9, 6.1 to 6.6, or 6.1 to 6.5. In certain embodiments, the pH of a composition comprising the diluted liposomal adjuvant and a chimeric influenza virus HA polypeptide described herein, or a composition thereof, is between 6 and 7, 6. 1 and 6.9, 6. 1 to 6.6, or 6. 1 to 6.5.

[00391] In certain embodiments, the liposomal adjuvant is a liposomal adjuvant described in U.S. Patent Application Publication No. 2008/0279926, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In certain embodiments, the liposomal adjuvant is ASOl as described in Section 6 below. In certain embodiments, the liposomal adjuvant is ASOlB-derived as described in Section 6 below. In certain embodiments, the liposomal adjuvant is ASOlE-like as described in Section 6 below. In certain embodiments, the liposomal adjuvant is ASOl as described in International Publication No. WO 2007/068907, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In certain embodiments, the liposomal adjuvant is ASOl as described in U.S. Patent Application Publication No. 2008/0279926, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In certain embodiments, the liposomal adjuvant is ASO IE as described in International Publication No. WO 2007/068907, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In certain embodiments, the liposomal adjuvant is ASO IE as described in U.S. Patent Application

Publication No. 2008/0279926, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In certain embodiments, the liposomal adjuvant is ASO IB as described in International Publication No. WO 2007/068907, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In certain embodiments, the liposomal adjuvant is ASO IB as described in U.S. Patent Application Publication No. 2008/0279926, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein.

[00392] In a specific embodiment, the liposomal adjuvant comprises MPL and QS-21 in a quenched form with cholesterol. In a specific embodiment, the liposomal adjuvant is made as described in International Publication No. WO 96/33739 or U.S. Patent No. 6,846,489, each of which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In specific embodiments, the liposomal adjuvant is prepared essentially as Example 1.1 of International Publication No. WO 96/33739 or U.S. Patent No. 6,846,489, each of, which is incorporated by reference herein in its entirety with respect to the compositions, methods, and kits described herein. In a specific embodiment, the liposomal adjuvant comprises DOPC, cholesterol, MPL, and QS21. In a specific embodiment, the liposomal adjuvant comprises DOPC, cholesterol, MPL, QS21, phosphate NaCl buffer, and water. In a specific embodiment, the liposomal adjuvant comprises 1000 μg of DOPC, 250 μg of cholesterol, 50 μg of MPL, 50 μ§Λ1θ8ε of QS21, phosphate NaCl buffer, and water to a volume of 0.5 mL (also referred to herein as "ASOl"). In a specific embodiment, the liposomal adjuvant comprises 500 μg of DOPC, 125 μg of cholesterol, 25 μg of MPL, 25

of QS21, phosphate NaCl buffer, and water to a volume of 0.5 mL (also referred to herein as "ASO IE").

[00393] In certain embodiments, the liposomes are 30-200 nm in size. In certain

embodiments, the liposomes are 95-120 nm in size.

[00394] In a specific embodiment, the liposomal adjuvant is one described in Section 6, infra. 5.9 PROPHYLACTIC AND THERAPEUTIC USES

[00395] In one aspect, provided herein are methods for inducing an immune response in a subject utilizing an active compound {e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), a vector {e.g., a viral vector) containing or expressing such a polypeptide(s), or a composition described herein). In a specific embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof an effective amount of an immunogenic composition described herein. In another embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof an effective amount of a nucleic acid sequence encoding a chimeric hemagglutinin (HA) polypeptide described herein, or an immunogenic composition thereof. In another embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof an effective amount of a viral vector containing or expressing a chimeric hemagglutinin (HA) polypeptide described herein, or an immunogenic composition thereof. In certain embodiments, a chimeric hemagglutinin (HA) polypeptide described herein used in the method is a purified chimeric hemagglutinin (HA) polypeptide described herein derived from a mammalian cell, a plant cell, or an insect cell.

[00396] In a specific embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof a subunit vaccine described herein. In another embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof a live virus vaccine described herein. In particular embodiments, the live virus vaccine comprises an attenuated virus. In another embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof an inactivated virus vaccine described herein. In another embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof a split virus vaccine described herein. In another embodiment, a method for inducing an immune response to an influenza virus hemagglutinin polypeptide in a subject comprises administering to a subject in need thereof a virus-like particle vaccine described herein. In another embodiment, a method for inducing an immune response to an influenza hemagglutinin polypeptide comprises administering to a subject in need thereof a virosome described herein. In certain embodiments, a chimeric hemagglutinin (HA) polypeptide described herein used in the method is a purified chimeric hemagglutinin (HA) polypeptide described herein derived from a mammalian cell, a plant cell, or an insect cell.

[00397] In one embodiment, provided herein is a method for immunizing against influenza virus in a subject, comprising administering to the subject an immunogenic composition described herein (e.g., in Section 5.8 above or Section 6 below). In some embodiments, the immunogenic composition comprises a chimeric HA polypeptide described herein (e.g., in Section 5.1 above or Section 6 below) and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). In a specific embodiment, the immunogenic composition comprises an inactivated influenza virus containing a chimeric HA polypeptide described herein and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). In another specific embodiment, the immunogenic composition comprises a split influenza virus and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below), wherein the split influenza virus comprises a chimeric HA polypeptide described herein.

[00398] In another aspect, provided herein is a method for immunizing against influenza virus in a subject, comprising administering to the subject an immunogenic composition described herein (e.g., in Section 5.8 above) and administering to the subject a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). In one embodiment, provided herein is a method for immunizing against influenza virus in a subject, comprising administering to the subject an immunogenic composition described herein (e.g., in Section 5.8 above) in combination with a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). The immunogenic composition may be administered to the subject concurrently with, prior to (e.g., less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 45 minutes, less than 60 minutes, less than 1.5 hours, or less than 2 hours prior to), or subsequent to (e.g., less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 45 minutes, less than 60 minutes, less than 1.5 hours, or less than 2 hours after) the administration of the liposomal adjuvant described herein. In a specific embodiment, the immunogenic composition and the liposomal adjuvant described herein are administered via the same route of administration. In other embodiments, the immunogenic composition and the liposomal adjuvant are administered via different routes of administration. In a specific embodiment, the immunogenic composition comprises an inactivated influenza virus containing a chimeric HA polypeptide described herein. In another specific embodiment, the immunogenic composition comprises a split influenza virus, wherein the split influenza virus comprises a chimeric HA polypeptide described herein. In some embodiments, the

immunogenic composition does not comprise an adjuvant. In other embodiments, the immunogenic composition comprises an adjuvant other than a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below).

[00399] In another embodiment, provided herein are immunization regimens involving a first immunization (e.g., priming) with a vaccine formulation described herein followed by one, two, or more additional immunizations (e.g., boostings) with a vaccine formulation. In a specific embodiment, the vaccine formulation used in the first immunization is the same type of vaccine formulation used in one, two or more additional immunizations. For example, if the vaccine formulation used in the first immunization is an inactivated influenza virus vaccine formulation, the vaccine formulation used for the one, two or more additional immunizations may be the same type of vaccine formulation, i.e., an inactivated influenza virus vaccine formulation. In other specific embodiments, the vaccine formulation used in the first immunization is different from the type of vaccine formulation used in one, two or more additional immunizations. For example, if the vaccine formulation used in the first immunization is a live influenza virus vaccine formulation, the vaccine formulation used in the one, two or more additional

immunizations is another type of vaccine formulation, such as an inactivated influenza virus. In another example, if the vaccine formulation used in the first immunization is a live attenuated influenza virus vaccine formulation, the vaccine formulation used in the one, two or more additional immunizations is another type of vaccine formulation, such as an inactivated influenza virus. In certain embodiments, the inactivated influenza virus is in a formulation comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In certain embodiments, the vaccine formulation used in the additional immunizations changes. For example, if a live attenuated influenza virus vaccine formulation is used for one additional immunization, then one or more additional immunizations may use a different vaccine formulation, such as an inactivated vaccine formulation. In a particular embodiment, a live influenza virus vaccine formulation is administered to a subject followed by an inactivated vaccine formulation (e.g., split virus vaccine or subunit vaccine). In specific embodiments, the inactivated vaccine formulation comprises a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In a specific embodiment, if a vaccine formulation used in an immunization regimen described herein comprises a chimeric HA, then the HA globular head domain of the chimeric HA changes with each immunization while the HA stem domain of the chimeric HA remains the same. In certain embodiments, the HA stem domain of the chimeric HAs are stem domains from an influenza virus HI or H3 subtype. In some embodiments, the influenza virus HA globular head domain of a chimeric HA used in one of the vaccine formulations is from an influenza A virus H4, H5, H7, H8, HI 1, H12, H14 or H15 subtype. [00400] In another embodiment, provided herein are immunization regimens involving a first immunization (e.g., priming) with a vaccine formulation described herein followed by one, two, or more additional immunizations (e.g., boostings) with a vaccine formulation. In a specific embodiment, the vaccine formulation used in the first immunization comprises 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 from an influenza virus one subtype (e.g., an H5 or H8 subtype) and the HA stem domain polypeptide is from a different subtype (e.g., an influenza virus HI or H3 subtype). In a specific

embodiment, the vaccine formulation used in the second immunization comprises an inactivated influenza virus comprising a second chimeric HA and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above), 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 from a different influenza virus subtype (e.g., an H5 or H8 subtype) and the HA stem domain polypeptide is the same as in the first chimeric HA. In certain embodiments, the globular head domain of the first chimeric HA is from an influenza virus H5 subtype and the globular head domain of the second chimeric HA is from an influenza virus H8 subtype. In other embodiments, the globular head domain of the first chimeric HA is from an influenza virus H8 subtype and the globular head domain of the second chimeric HA is from an influenza virus H5 subtype. In a specific embodiment, the period of time between the first immunization and the second immunization is 6 weeks, 8 weeks, 9 weeks, 12 weeks, 3 months, 4 months, 5 months or 6 months. In a specific embodiment, the period of time between the first immunization and the second immunization is about 6 weeks, about 8 weeks, about 12 weeks, about 3 months, about 4 months, about 6 months, or about 9 months. In another specific embodiment, the period of time between the first immunization and second immunization is 1 week to 9 months, 3 weeks to 8 months, 6 weeks to 12 weeks, 4 weeks to 6 months, 5 weeks to 5 months, 6 weeks to 4 months, 7 weeks to 4 months, 8 weeks to 4 months, 8 weeks to 3 months, 3 months to 6 months, 3 months to 9 months, or 6 months to 9 months.

[00401] In a specific embodiment, 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 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 (e.g., 1-6 months, 3-6 months, 6-9 months, 3-9 months, or 9-12 months), 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. In specific embodiments, the inactivated influenza virus is a influenza virus split virus vaccine comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In specific embodiments, the inactivated influenza virus is an influenza virus subunit vaccine comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above).

[00402] In a specific embodiment, provided herein is an immunization regimen comprising administering to a subject (i) a first vaccine formulation comprising a chimeric HA comprising an influenza virus HA globular head domain from an influenza A virus H8 subtype and an influenza virus HA stem domain from an influenza A virus HI subtype; (ii) a second vaccine formulation comprising a chimeric HA comprising an influenza virus HA globular head domain from an influenza A virus H5 subtype and an influenza virus HA stem domain from an influenza A virus HI subtype; and (iii) a third vaccine formulation comprising a chimeric HA comprising an influenza virus HA globular head domain from an influenza A virus HI 1 subtype and an influenza virus HA stem domain from an influenza A virus HI subtype. In a specific

embodiment, the influenza virus HA stem domain for the three chimeric HAs is from the same influenza virus HI subtype. The order of the vaccine formulations may vary. For example, the first vaccine formulation may be administered first, second, or third, the second vaccine formulation may be administered first, second, or third, and the third vaccine formulation may be administered first, second, or third. In certain embodiments, the first, second, and third vaccine formulations further comprise a chimeric HA, wherein the chimeric HA comprises an influenza virus HA globular head domain from an influenza virus H4, H14, HI 5, or H7 subtype and an influenza virus stem domain from an influenza virus H3 subtype. For example, the first vaccine may further comprise a second chimeric HA, wherein the second chimeric HA comprises an influenza virus HA globular head domain from an influenza A virus H4 subtype and an influenza virus HA stem domain from an influenza A virus H3 subtype. The combinations of the first chimeric HA comprising a stem domain from an influenza A virus HI subtype and the second chimeric HA comprising a stem domain from an influenza A virus H3 subtype in the vaccine formulation may vary. In a specific embodiment, one, two, or all of the first, second, and third vaccines may comprise a liposomal adjuvant described herein (e.g., described in Section 5.8.5 above or Section 6 below). In some embodiments, one, two, or all of the first, second, and third vaccines are inactivated influenza virus vaccines. In a specific embodiment, one, two, or all of the first, second, and third vaccines are subunit vaccines. In another specific embodiment, one, tow, or all of the first, second, and third vaccines are split virus vaccines. In a specific embodiment, the second vaccine formulation is administered 1 week to 9 months, 3 weeks to 8 months, 6 weeks to 12 weeks, 4 weeks to 6 months, 5 weeks to 5 months, 6 weeks to 4 months, 7 weeks to 4 months, 8 weeks to 4 months, 8 weeks to 3 months, 3 months to 6 months, 3 months to 9 months, or 6 months to 9 months after the first vaccine formulation is administered. In a specific embodiment, the third vaccine formulation is administered 1 week to 9 months, 3 weeks to 8 months, 6 weeks to 12 weeks, 4 weeks to 6 months, 5 weeks to 5 months, 6 weeks to 4 months, 7 weeks to 4 months, 8 weeks to 4 months, 8 weeks to 3 months, 3 months to 6 months, 3 months to 9 months, or 6 months to 9 months after the second vaccine formulation is administered.

[00403] In a specific embodiment, 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 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 (e.g., 1-6 months, 3-6 months, 6-9 months, 3-9 months, or 9-12 months), 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. In specific embodiments, the inactivated influenza virus is a influenza virus split virus vaccine comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In specific embodiments, the inactivated influenza vims is an influenza virus subunit virus vaccine comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above).

[00404] In another specific embodiment, provided herein is an immunization regimen comprising (i) administering to a subject a live-attenuated influenza virus, wherein said live- attenuated influenza virus comprises or is engineered to encode, or both, a first chimeric HA, wherein the first chimeric HA comprises an influenza virus HA globular head domain from a first influenza A virus subtype (e.g. , H5 or H8) and an influenza virus HA stem domain from an influenza A virus HI subtype, and (ii) subsequently administering to the subject a vaccine formulation comprising a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above) and an inactivated influenza virus, wherein the inactivated influenza virus comprises a second chimeric HA, wherein the second chimeric HA comprises an influenza virus HA globular head domain from a second influenza A virus subtype (e.g., H5 or H8) and the influenza virus HA stem domain. In a specific embodiment, the first influenza A virus is an H5 subtype and the second influenza A virus is an H8 subtype. In another specific embodiment, the first influenza A virus is an H8 and the second influenza A virus is an H5. In a specific embodiment, the liposomal adjuvant is AS01 (see, e.g., Section 5.8.5 above and Section 6 below). In a specific embodiment, the vaccine formulation is administered 1 week to 9 months, 3 weeks to 8 months, 6 weeks to 12 weeks, 4 weeks to 6 months, 5 weeks to 5 months, 6 weeks to 4 months, 7 weeks to 4 months, 8 weeks to 4 months, 8 weeks to 3 months, 3 months to 6 months, 3 months to 9 months, or 6 months to 9 months after the live-attenuated influenza virus is administered.

[00405] In specific embodiments, provided herein is a method of immunizing a subject against influenza virus, comprising: (a) administering to the subject a live attenuated influenza virus; and (b) after a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 3-9 months, 9-12 months, etc.) administering to the subject an immunogenic composition comprising chimeric HA described herein and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In a specific embodiment, the stem domain of the hemagglutinin of the live attenuated influenza virus administered in step (a) is the same subtype or strain as the stem domain polypeptide of the chimeric HA administered in step (b), and the globular head domain of the hemagglutinin of the live attenuated influenza virus administered in step (a) is

heterologous to the globular head domain of the chimeric HA used in step (b). In certain embodiments, the method further comprises step (c), which comprises administering to the subject one or more additional vaccine formulations described herein a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) after step (b). In certain embodiments, the one or more additional vaccine formulations comprise a chimeric HA, or a vector comprising the same, and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In a specific embodiment, the stem domain of the hemagglutinin of the live attenuated influenza virus administered in step (a) and the stem domain polypeptide of the chimeric HA in step (b) are the same subtype or strain as the stem domain polypeptide of the chimeric HA administered in step (c), and, the chimeric HA is utilized in step (c), the globular head domain of the hemagglutinin of the live attenuated influenza virus administered in step (a) and the globular head domain of the chimeric HA administered in step (b) are heterologous to the globular head domain of the chimeric HA used in step (c). In a specific embodiment, the one or more additional vaccine formulations comprises an inactivated influenza virus vector comprising the same and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In a specific embodiment, the live attenuated influenza virus and/or inactivated influenza virus are administered to the subject intranasally. In certain embodiments, the attenuated influenza virus and/or inactivated influenza virus are administered to the subject intramuscularly or subcutaneously.

[00406] In specific embodiments, provided herein is a method of immunizing a subject against influenza virus, comprising: (a) administering to the subject an inactivated influenza virus; and (b) after a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) administering to the subject an immunogenic composition comprising a chimeric HA described herein and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In a specific embodiment, the stem domain of the hemagglutinin of the inactivated influenza virus administered in step (a) is the same subtype or strain as the stem domain polypeptide of the chimeric HA administered in step (b), and the globular head domain of the hemagglutinin of the inactivated influenza virus administered in step (a) is heterologous to the globular head domain of the chimeric HA used in step (b). In certain embodiments, the method comprises step (c), which comprises administering to the subject one or more additional vaccine formulations described herein a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) after step (b). In certain embodiment, the one or more additional vaccine formulations comprise a chimeric HA, or vector comprising the same, and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In a specific embodiment, the stem domain of the hemagglutinin of the inactivated influenza virus administered in step (a) and the stem domain polypeptide of the chimeric HA administered in step (b) are the same subtype or strain as the stem domain polypeptide of the chimeric HA administered in step (c), and, the chimeric HA is utilized in step (c), the globular head domain of the hemagglutinin of the inactivated influenza virus administered in step (a) and the globular head domain of the chimeric HA administered in step (b) are heterologous to the globular head domain of the chimeric HA used in step (c). In a specific embodiment, the one or more additional vaccine formulations comprises an inactivated influenza virus or live attenuated influenza virus and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above). In a specific embodiment, the inactivated influenza virus and/or inactivated influenza virus are administered to the subject intranasally. In certain embodiments, the attenuated influenza virus and/or inactivated influenza virus are administered to the subject intramuscularly or subcutaneously.

[00407] In one embodiment, provided herein is a method of immunizing a subject against influenza virus, comprising: (a) administering to the subject a first inactivated influenza virus vaccine comprising a first chimeric HA described herein; and (b) after a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) administering to the subject a second inactivated influenza virus vaccine comprising a second chimeric HA described herein and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above or Section 6 below). In a specific embodiment, the first chimeric HA used in step (a) comprises a different HA globular head domain than the second chimeric HA used in step (b). In specific embodiments, the stem domain of the first and second chimeric HAs are the same (e.g., from the same influenza A virus strain). In a specific embodiment, the first inactivated influenza virus vaccine comprises a liposomal adjuvant described herein (e.g., a liposomal adjuvant described in Section 5.8.5 above or Section 6 below). In some embodiments, the first and second inactivated influenza virus vaccines are both subunit vaccines. In other embodiments, the first and second inactivated virus vaccines are split virus vaccines. In yet other embodiments, the first and second inactivated virus vaccines are inactivated whole virus vaccines. In certain embodiments, the first inactivated influenza virus vaccine is a different type of inactivated vaccine than the second inactivated influenza virus vaccine. In certain embodiments, the method further comprises administering to the subject one or more additional vaccine formulations described herein a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) after step (b). In a specific embodiment, the method comprises administering the subject one or more additional inactivated influenza virus vaccine formulations described herein a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) after step (b). In a specific embodiment, the first and second inactivated influenza virus vaccines are administered to the subject intranasally or subcutaneously. In some embodiments, one or more additional vaccine formulations may also be administered intranasally or subcutaneously, In certain embodiments, the first and second inactivated influenza virus vaccines are administered intramuscularly. In some embodiments, the one or more additional vaccine formulations may also be administered intramuscularly.

[00408] In one embodiment, provided herein is a method of immunizing a subject against influenza virus, comprising: (a) administering to the subject a first inactivated influenza virus vaccine comprising a first chimeric HA described herein and a liposomal adjuvant (e.g., a liposomal adjuvant described in Section 5.8.5 above or Section 6 below); and (b) after a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) administering to the subject a second inactivated influenza virus vaccine comprising a second chimeric HA described herein. In a specific embodiment, the first chimeric HA used in step (a) comprises a different HA globular head domain than the second chimeric HA used in step (b). In specific embodiments, the stem domain of the first and second chimeric HAs are the same (e.g., from the same influenza A virus strain). In a specific embodiment, the second inactivated influenza virus vaccine comprises a liposomal adjuvant described herein (e.g., a liposomal adjuvant described in Section 5.8.5 above or Section 6 below). In some embodiments, the first and second inactivated influenza virus vaccines are both subunit vaccines. In other

embodiments, the first and second inactivated virus vaccines are split virus vaccines. In yet other embodiments, the first and second inactivated virus vaccines are inactivated whole virus vaccines. In certain embodiments, the first inactivated influenza virus vaccine is a different type of inactivated vaccine than the second inactivated influenza virus vaccine. In certain

embodiments, the method further comprises administering to the subject one or more additional vaccine formulations described herein a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) after step (b). In a specific embodiment, the method comprises administering the subject one or more additional inactivated influenza virus vaccine formulations described herein a certain period of time (e.g., 1-6 months, 3-6 months, 6-9 months, 6-9 months, 9-12 months, etc.) after step (b). In a specific embodiment, the first and second inactivated influenza virus vaccines are administered to the subject intranasally or

subcutaneously. In some embodiments, one or more additional vaccine formulations may also be administered intranasally or subcutaneously, In certain embodiments, the first and second inactivated influenza virus vaccines are administered intramuscularly. In some embodiments, the one or more additional vaccine formulations may also be administered intramuscularly.

[00409] In specific embodiments, provided herein is a method for immunizing a subject against influenza virus comprising: (A) administering to the subject a first immunogenic composition comprising a first chimeric HA polypeptide, a second chimeric HA polypeptide and a third chimeric HA polypeptide, wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of a first influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the first influenza B virus HA globular head domain, and the third HA stem domain comprises the HA stem domain of the first influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain; (B) administering to the subject a second immunogenic composition comprising a fourth chimeric HA polypeptide, a fifth chimeric HA polypeptide and a sixth chimeric HA polypeptide, wherein (i) the fourth chimeric HA polypeptide comprises a fourth HA globular head domain of a fifth influenza A virus of a fifth HA subtype and a fourth HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the fourth HA globular head domain is heterologous to the fourth HA stem domain, (ii) the fifth chimeric HA polypeptide comprises a fifth HA globular head domain of a sixth influenza A virus of a sixth HA subtype and a fifth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the fifth HA globular head domain is heterologous to the fifth HA stem domain, wherein the second, fourth, fifth and sixth HA subtypes are different from each other, and (iii) the sixth chimeric HA polypeptide comprises a sixth HA globular head domain and a sixth HA stem domain, wherein the sixth HA globular head domain comprises the HA globular head domain of a second influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the second influenza B virus HA globular head domain, and the sixth HA stem domain comprises the HA stem domain of the second influenza B virus, wherein the sixth HA globular head domain is heterologous to the sixth HA stem domain; and (C) administering to the subject a third immunogenic composition comprising a seventh chimeric HA polypeptide, an eighth chimeric HA polypeptide and a ninth chimeric HA polypeptide, wherein (i) the seventh chimeric HA polypeptide comprises a seventh HA globular head domain of a seventh influenza A virus of a seventh HA subtype and a seventh HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the seventh HA globular head domain is heterologous to the seventh HA stem domain, (ii) the eighth chimeric HA polypeptide comprises an eighthHA globular head domain of an eighth influenza A virus of an eighth HA subtype and an eighth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the eighth HA globular head domain is heterologous to the eighth HA stem domain, wherein the second, fourth, seventh and eighth HA subtypes are different from each other, and (iii) the ninth chimeric HA polypeptide comprises a ninth HA globular head domain and a ninth HA stem domain, wherein the ninth HA globular head domain comprises the HA globular head domain of a third influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the third influenza B virus HA globular head domain, and the nighth HA stem domain comprises the HA stem domain of the third influenza B virus, wherein the ninth HA globular head domain is heterologous to the ninth HA stem domain. Either one, two or all of the first, second and third immunogenic compositions may comprise an adjuvant (e.g., a liposomal adjuvant such as described in Section 5.8.5 above or Section 6 below). The immunogenic composition may be an inactivated vaccine, such as subunit vaccine, split vaccine or whole inactivated virus vaccine. In particular embodiments, the first, second, fourth, fifth, seventh, and eighth chimeric HA polypeptides may be ones described in Section 5.1 above (in particular, Section 5.1.1.1 above), Section 6.1 below, Section 6.2 below, Section 6.3 below, or Section 6.4 below and the third, sixth and ninth chimeric HA polypeptide may be one described in Section 5.1.2 above or Section 6.5 below. In certain embodiments, the first and second immunogenic compositions are administered 1-6 months, 6-9 months, 3-6 monhts, 3-9 months, or 9-12 monhts apart. In some embodiments, the second and third immunogenic compositions are administered 1-6 months, 6-9 months, 3-9 months, or 9-12 months apart. In specific embodiments, the time period between administration of each of the immunogenic compositions is 1-6 months, 6-9 months, 3-9 months, or 9-12 months. In a specific embodiment, the second and fourth HA subtypes are HI and H3, respectively. See, e.g,. Sections 5.1 and 6 for examples of chimeric HA polypeptides that may be used in such immunogenic compositions.

[00410] In specific embodiments, a method for immunizing a subject against influenza virus comprising: (A) administering to the subject a first immunogenic composition comprising a first chimeric HA polypeptide, a second chimeric HA polypeptide, a third chimeric HA polypeptide and an adjuvant (in specific embodiments, a liposomal adjuvant such as described in such as described in Section 5.8.5 above or Section 6 below), wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of a first influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the first influenza B virus HA globular head domain, and the third HA stem domain comprises the HA stem domain of the first influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain; (B) administering to the subject a second immunogenic composition comprising a fourth chimeric HA polypeptide, a fifth chimeric HA polypeptide, a sixth chimeric HA polypeptide and an adjuvant (in specific embodiments, a liposomal adjuvant such as described in such as described in Section 5.8.5 above or Section 6 below), wherein (i) the fourth chimeric HA polypeptide comprises a fourth HA globular head domain of a fifth influenza A virus of a fifth HA subtype and a fourth HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the fourth HA globular head domain is heterologous to the fourth HA stem domain, (ii) the fifth chimeric HA polypeptide comprises a fifth HA globular head domain of a sixth influenza A virus of a sixth HA subtype and a fifth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or H15), wherein the fifth HA globular head domain is heterologous to the fifth HA stem domain, wherein the second, fourth, fifth and sixth HA subtypes are different from each other, and (iii) the sixth chimeric HA polypeptide comprises a sixth HA globular head domain and a sixth HA stem domain, wherein the sixth HA globular head domain comprises the HA globular head domain of a second influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the second influenza B virus HA globular head domain, and the sixth HA stem domain comprises the HA stem domain of the second influenza B virus, wherein the sixth HA globular head domain is heterologous to the sixth HA stem domain; and (C) administering to the subject a third immunogenic composition comprising a seventh chimeric HA polypeptide, an eighth chimeric HA polypeptide, a ninth chimeric HA polypeptide and an adjuvant (in specific embodiments, a liposomal adjuvant such as described in Section 5.8.5 above or Section 6 below), wherein (i) the seventh chimeric HA polypeptide comprises a seventh HA globular head domain of a seventh influenza A virus of a seventh HA subtype and a seventh HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1 (e.g., HI, H2, H5, H6, H8, H9, HI 1, H12, H13, or H16), and wherein the seventh HA globular head domain is heterologous to the seventh HA stem domain, (ii) the eighth chimeric HA polypeptide comprises an eighth HA globular head domain of an eighth influenza A virus of an eighth HA subtype and an eighth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2 (e.g., H3, H4, H7, H10, H14, or HI 5), wherein the eighth HA globular head domain is heterologous to the eighth HA stem domain, wherein the second, fourth, seventh and eighth HA subtypes are different from each other, and (iii) the ninth chimeric HA polypeptide comprises a ninth HA globular head domain and a ninth HA stem domain, wherein the ninth HA globular head domain comprises the HA globular head domain of a third influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the third influenza B virus HA globular head domain, and the nighth HA stem domain comprises the HA stem domain of the third influenza B virus, wherein the ninth HA globular head domain is heterologous to the ninth HA stem domain. The immunogenic composition may be an inactivated vaccine, such as subunit vaccine, split vaccine or whole inactivated virus vaccine. In particular embodiments, the first, second, fourth, fifth, seventh, and eighth chimeric HA polypeptides may be ones described in Section 5.1 above (in particular, Section 5.1.1.1 above), Section 6.1 below, Section 6.2 below, Section 6.3 below, or Section 6.4 below and the third, sixth and ninth chimeric HA polypeptide may be one described in Section 5.1.2 above or Section 6.5 below. In certain embodiments, the first and second immunogenic compositions are administered 1-6 months, 6-9 months, 3-6 monhts, 3-9 months, or 9-12 monhts apart. In some embodiments, the second and third immunogenic compositions are administered 1-6 months, 6-9 months, 3-9 months, or 9-12 months apart. In specific embodiments, the time period between administration of each of the immunogenic compositions is 1-6 months, 6-9 months, 3-9 months, or 9-12 months. In a specific embodiment, the second and fourth HA subtypes are HI and H3, respectively. See, e.g,. Sections 5.1 and 6 for examples of chimeric HA polypeptides that may be used in such immunogenic compositions.

[00411] In another aspect, provided herein is a method of immunizing a subject against influenza virus comprising administering an immunogenic composition comprising a chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain, wherein the globular head domain is heterologous to the stem domain, wherein the liposomal adjuvant comprises liposomes and either a TLR4-4 agonist, saponin or both, and wherein the composition in an animal induces influenza virus-specific T cells in the lung. See Section 5.1 above and Section 6 below for examples of chimeric HA polypeptides that may be present in the composition. In a particular embodiment, the liposomal adjuvant is the adjuvant described in Section 5.8.5 above or Section 6 below.

[00412] In another aspect, provided herein is an immunogenic composition comprising a chimeric influenza virus HA polypeptide and a liposomal adjuvant, for use in a method of immunizing a subject against influenza virus, wherein the method comprises administering the chimeric HA polypeptide to the subject, wherein chimeric HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain, wherein the globular head domain is heterologous to the stem domain, wherein the liposomal adjuvant comprises liposomes and either a TLR4-4 agonist, saponin or both, and wherein the composition in an animal induces influenza virus-specific T cells in the lung. See Section 5.1 above and Section 6 below for examples of chimeric HA polypeptides that may be present in the composition. In a particular embodiment, the liposomal adjuvant is the adjuvant described in Section 5.8.5 above or Section 6 below.

[00413] In some embodiments, the immune response induced by an immunogenic

composition described herein is effective to prevent and/or treat an influenza virus infection caused by one, two, three, four or five subtypes of influenza A virus and/or one or both lineages of influenza B virus. In certain embodiments, the immune response induced by an immunogenic composition described herein is effective to prevent and/or treat an influenza virus infection caused by six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen subtypes of influenza A virus and/or one or both lineages of influenza B virus. In some embodiments, the immune response induced by an immunogenic composition described herein is effective to prevent and/or treat an influenza virus infection caused by one or more variants within the same subtype of influenza A virus or within the same lineage of influenza B virus.

[00414] In some embodiments, the immune response induced by an immunogenic

composition described herein is effective to prevent and/or treat an influenza virus infection caused by both HlNl and H2N2 subtypes. In other embodiments, the immune response induced by an immunogenic composition described herein is not effective to prevent and/or treat an influenza virus infection caused by both HlNl and H2N2 subtypes. In some embodiments, the immune response induced by an immunogenic composition described herein is effective to prevent and/or treat an influenza virus infection caused by HlNl, H2N2, and H3N2 subtypes. In some embodiments, the immune response induced by an immunogenic composition described herein is effective to prevent and/or treat an influenza virus infection caused by H3N2 subtypes. In other embodiments, the immune response induced by an immunogenic composition described herein is not effective to prevent and/or treat an influenza virus infection caused by H3N2 subtypes.

[00415] In some embodiments, the immune response induced by an immunogenic

composition described herein is effective to prevent and/or treat an influenza virus disease caused by any subtype or strain of influenza virus. In certain embodiments, the immune response induced by an immunogenic composition described herein is effective to prevent and/or treat an influenza virus disease caused by a subtype of influenza virus that belongs to one HA group and not the other HA group. For example, the immune response induced may be effective to prevent and/or treat an influenza virus disease caused by an influenza virus that belongs to the HA group consisting of HI 1, H13, H16, H9, H8, H12, H6, HI, H5 and H2. Alternatively, the immune response induced may be effective to prevent and/or treat an influenza virus disease caused by an influenza virus that belongs to the HA group consisting of H3, H4, H14, H10, H15 and H7. In some embodiments, the immune response induced by an immunogenic composition described herein is effective to prevent and/or treat an influenza virus disease caused by any of one, two, three, four or five subtypes of influenza virus. In certain embodiments, the immune response induced by an active compound or a composition described herein is effective to prevent and/or treat an influenza virus disease caused by any of six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen subtypes of influenza virus. In some embodiments, the immune response induced by an active compound or a composition described herein is effective to prevent and/or treat an influenza virus disease caused by one or more variants within the same subtype of influenza virus.

[00416] In some embodiments, the immune response induced by an immunogenic

composition described herein is effective to reduce symptoms resulting from an influenza virus disease/infection. Symptoms of influenza vims disease/infection include, but are not limited to, body aches (especially joints and throat), fever, nausea, headaches, irritated eyes, fatigue, sore throat, reddened eyes or skin, and abdominal pain.

[00417] In some embodiments, the immune response induced by an immunogenic

composition described herein is effective to reduce the hospitalization of a subject suffering from an influenza virus disease/infection. In some embodiments, the immune response induced by an immunogenic composition described herein is effective to reduce the duration of hospitalization of a subject suffering from an influenza virus disease/infection.

[00418] In certain embodiments, the methods for immunizing against influenza virus in a subject (e.g., a human or non-human animal) provided herein result in a hemagglutination inhibition ("HI") antibody titer at least 1 AO. In certain embodiments, the methods for immunizing against influenza virus in a subject (e.g., a human or non-human animal) provided herein result in seroconversion. In certain embodiments, seroconversion is when a subject has (a) an HI titer of less than 1 : 10 prior to being administered an immunogenic composition described herein, and (b) an HI titer of at least 1 AO and a minimum four-fold rise in HI antibody titer after being administered the immunogenic composition described herein. In certain embodiments, the methods for immunizing against influenza virus in a subject (e.g., a human or non-human animal) provided herein result in the lower bound of a two-sided 95% confidence interval ("CI") for the percent of subjects receiving the immunogenic composition(s) of the methods achieving seroconversion for HI antibody meeting or exceeding 40%, wherein the subjects are less than 65 years of age. In certain embodiments, the methods for immunizing against influenza virus in a subject (e.g., a human or non-human animal) provided herein result in the lower bound of a two-sided 95% CI for the percent of subjects receiving the immunogenic composition(s) of the methods achieving an HI antibody titer of at least 1 :40 meeting or exceeding 70%, wherein the subjects are less than 65 years of age. In certain embodiments, the methods for immunizing against influenza virus in a subject (e.g., a human or non-human animal) provided herein result in the lower bound of a two-sided 95% CI for the percent of subjects receiving the immunogenic composition(s) of the methods achieving seroconversion for HI antibody meeting or exceeding 30%, wherein the subjects are at least 65 years of age. In certain embodiments, the methods for immunizing against influenza virus in a subject (e.g., a human or non-human animal) provided herein result in the lower bound of a two-sided 95% CI for the percent of subjects receiving the immunogenic composition(s) of the method achieving an HI antibody titer of at least 1 :40 meeting or exceeding 60%, wherein the subjects are at least 65 years of age. Techniques for determining HI antibody titer are known to one skilled in the art. Methods for evaluating the lower bound of the two-sided 95% CI are known to one skilled in the art. See, e.g., the U.S. Food & Drug Administration Guidance for Industry: Clinical Data Needed to Support the Licensure of Pendemic Influenza Vaccines and Guideance for Industry: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines. In specific embodiments, a method of immunization described herein meet one, two, three, or more criteria for demonstrating effectiveness of an influenza vaccine as determined according to the U. S. Food & Drug Administration Guidance for Industry: Clinical Data Needed to Support the Licensure of Pendemic Influenza Vaccines or Guideance for Industry: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines.

[00419] In a specific embodiment, the presence of a liposomal adjuvant described herein {e.g., in Section 5.8.5 above or Section 6 below) in an inactivated vaccine {e.g., a split virus vaccine, an inactivated whole virus vaccine, or a subunit vaccine) comprising a chimeric HA polypeptide described herein results in a higher amount of IgGi, IgG₃, or both, detected in subjects than the amount of IgGi, IgG₃, or both, detected in subjects vaccinated with the same chimeric HA vaccine composition without an adjuvant. In another specific embodiment, the present of a liposomal adjuvant described herein {e.g., in Section 5.8.5 above or Section 6 below) in an inactivated vaccine {e.g., a split virus vaccine, an inactivated whole virus vaccine, or a subunit vaccine) comprising a chimeric HA polypeptide described herein results in a higher ratio of IgG_2a to IgGi in mice when administered intramuscularly relative to mice administered the same vaccine without the adjuvant. In another specific embodiment, the presence of a liposomal adjuvant described herein {e.g., in Section 5.8.5 above or Section 6 below) in an immunogenic composition described herein has one or more of the effects described in Section 6 below for AS01, ASO IB, or ASOlE-like {e.g., the liposomal adjuvant in the compositions results in a similar effect on influenza virus-specific T cells in the lungs or on IgG_2a or IgGi levels as compared to the effects of an immunogenic composition without the adjuvant).

[00420] In another aspect, provided herein are methods for preventing, treating, or preventing and treating an influenza virus infection in a subject utilizing an immunogenic composition described herein. In a specific embodiment, a method for preventing or treating an influenza virus infection in a subject comprises administering to a subject in need thereof a subunit vaccine, a live virus vaccine, an inactivated virus vaccine, a split virus vaccine or a virus-like particle vaccine described herein. In a specific embodiment, a method for preventing or treating an influenza virus disease in a subject comprises administering to a subject in need thereof a subunit vaccine described herein. In another embodiment, a method for preventing or treating an influenza virus disease in a subj ect comprises administering to a subject in need thereof a live virus vaccine described herein. In particular embodiments, the live virus vaccine comprises an attenuated virus. In another embodiment, a method for preventing or treating an influenza virus disease in a subject comprises administering to a subject in need thereof an inactivated virus vaccine described herein. In another embodiment, a method for preventing or treating an influenza virus disease in a subj ect comprises administering to a subject in need thereof a split virus vaccine described herein. In another embodiment, a method for preventing or treating an influenza virus disease comprises administering to a subject in need thereof a virus-like particle vaccine described herein. In another embodiment, a method for preventing or treating an influenza virus disease in a subj ect, comprising administering to a subject in need thereof a virosome described herein.

[00421] In one embodiment, provided herein is a method for preventing influenza virus disease in a subject, comprising administering to the subject an immunogenic composition described herein (e.g., in 5.8.5 above or Section 6 below). In some embodiments, the immunogenic composition comprises a chimeric HA polypeptide described herein (e.g., in Section 5.1 above or Section 6 below) and a liposomal adjuvant described herein (e.g., in 5.8.5 above or Section 6 below). In a specific embodiment, the immunogenic composition comprises an inactivated influenza virus containing a chimeric HA polypeptide described herein and a liposomal adjuvant described herein (e.g., in 5.8.5 above or Section 6 below). In another specific embodiment, the immunogenic composition comprises a split influenza virus and a liposomal adjuvant described herein (e.g., in 5.8.5 above or Section 6 below), wherein the split influenza virus comprises a chimeric HA polypeptide described herein.

[00422] In another aspect, provided herein is a method for preventing influenza virus disease in a subject, comprising administering to the subject an immunogenic composition described herein (e.g., in Section 5.8 above) and administering to the subject a liposomal adjuvant described herein (e.g., in 5.8.5 above or Section 6 below). In one embodiment, provided herein is a method for immunizing against influenza virus in a subject, comprising administering to the subject an immunogenic composition described herein (e.g., in Section 5.8 above) in

combination with a liposomal adjuvant described herein (e.g., in 5.8.5 above or Section 6 below). The immunogenic composition may be administered to the subject concurrently with, prior to (e.g., less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 45 minutes, less than 60 minutes, less than 1.5 hours, or less than 2 hours prior to), or subsequent to (e.g., less than 5 minutes, less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 45 minutes, less than 60 minutes, less than 1.5 hours, or less than 2 hours after) the administration of the liposomal adjuvant described herein. In a specific embodiment, the immunogenic composition and the liposomal adjuvant described herein are administered via the same route of administration. In other embodiments, the immunogenic composition and the liposomal adjuvant are administered via different routes of administration. In a specific embodiment, the immunogenic composition comprises an inactivated influenza virus containing a chimeric HA polypeptide described herein. In another specific embodiment, the immunogenic composition comprises a split influenza virus, wherein the split influenza virus comprises a chimeric HA polypeptide described herein. In some embodiments, the immunogenic

composition does not comprise an adjuvant. In other embodiments, the immunogenic

composition comprises an adjuvant other than a and a liposomal adjuvant described herein (e.g., in 5.8.5 above or Section 6 below).

[00423] In certain embodiments, the methods for preventing or treating an influenza virus disease or infection in a subject (e.g., a human or non-human animal) provided herein result in a reduction in the replication of the influenza virus in the subject as measured by in vivo and in vitro assays known to those of skill in the art and described herein. In some embodiments, the replication of the influenza virus is reduced by 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 9 logs, or 8 to

9 logs. In a specific embodiment, the methods for preventing or treating an influenza virus disease or infection in a subject results in one, two, or more of the following: reduces the number of symptoms of the disease or infection, reduces the severity of the symptoms of the disease or infection, reduces the length of the disease or infection, or reduces hospitalization or

complications resulting from the disease or infection.

[00424] In a specific embodiment, a method of immunizing a subject against influenza virus comprises the methodology set forth in Section 6, infra. In another specific embodiment, a method for preventing an influenza virus disease in a subject comprises the methodology set forth in Section 6, infra. In another specific embodiment, a method of immunizing a subject against influenza virus comprises an immunization regimen set forth in Section 6, infra, using the same chimeric HA polypeptides or other chimeric HA polypeptides described herein. In another specific embodiment, a method for preventing an influenza virus disease in a subject comprises an immunization regimen set forth in Section 6, infra, using the same chimeric HA polypeptides or other chimeric HA polypeptides described herein.

[00425] Example 9 of International Publication No. WO 2013/043729 or U.S. Patent

Application Publication No. 2015/0132330, each of which is incorporated herein by reference in its entirety, sets forth how chimeric influenza virus HA polypeptides may be used to vaccinate subjects against influenza virus infection.

5.9.1 Combination therapies

[00426] In various embodiments, a chimeric influenza virus hemagglutinin polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such polypeptide(s), may be administered to a subject in combination with one or more other therapies (e.g., an antiviral, antibacterial, or immunomodulatory therapies). In some embodiments, a pharmaceutical composition (e.g., an immunogenic composition) described herein may be administered to a subject in combination with one or more therapies. The one or more other therapies may be beneficial in the treatment or prevention of an influenza virus disease or may ameliorate a symptom or condition associated with an influenza virus disease. In some embodiments, the one or more other therapies are pain relievers, anti-fever medications, or therapies that alleviate or assist with breathing. In certain embodiments, the therapies are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. In specific embodiments, two or more therapies are administered within the same patent visit.

5.9.2 Patient Populations

[00427] In certain embodiments, an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such polypeptide(s)), or composition described herein may be administered to a naive subject, i.e., a subject that does not have a disease caused by influenza virus infection or has not been and is not currently infected with an influenza virus infection. In one embodiment, an active compound or composition described herein is administered to a naive subject that is at risk of acquiring an influenza virus infection. In one embodiment, an active compound or composition described herein is administered to a subject that does not have a disease caused by the specific influenza virus, or has not been and is not infected with the specific influenza virus to which the chimeric hemagglutinin (HA) polypeptide induces an immune response. An active compound or composition described herein may also be administered to a subject that is and/or has been infected with the influenza virus or another type, subtype/lineage or strain of the influenza virus to which the chimeric hemagglutinin (HA) polypeptide induces an immune response.

[00428] In certain embodiments, an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s)), or composition described herein is administered to a patient who has been diagnosed with an influenza virus infection. In some embodiments, an active compound or composition described herein is administered to a patient infected with an influenza virus before symptoms manifest or symptoms become severe (e.g., before the patient requires hospitalization). In some

embodiments, an active compound or composition described herein is administered to a patient that is infected with or has been diagnosed with a different type, subtype/lineage, or strain of influenza virus than that of the influenza virus from which the immunodominant epitopes of the head domain of the chimeric hemagglutinin (HA) polypeptide of the active compound or composition was derived.

[00429] In some embodiments, a subject to be administered an active compound (e.g., a chimeric hemagglutinin (HA) described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s)) or composition described herein is an animal. In certain embodiments, the animal is a bird. In certain embodiments, the animal is a canine. In certain embodiments, the animal is a feline. In certain embodiments, the animal is a horse. In certain embodiments, the animal is a cow. In certain embodiments, the animal is a mammal, e.g., a horse, swine, mouse, or primate, preferably a human.

[00430] In specific embodiments, a subject administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s)) or composition described herein is a human infant. As used herein, the term "human infant" refers to a newborn to 1 year old human. In specific embodiments, a subject administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s)) or composition described herein is a human child. As used herein, the term "human child" refers to a human that is 1 year to 18 years old. In specific embodiments, a subject administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s)) or composition described herein is a human adult. In specific embodiments, a subject administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s)) or composition described herein is an elderly human.

[00431] In specific embodiments, a subject to be administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) containing or expressing such a polypeptide(s)), or composition described herein has one, two, or more characteristics in the inclusion criteria in the clinical study described in Section 6.4 below. In specific embodiments, a subject to be administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) containing or expressing such a polypeptide(s)), or composition described herein does not have one, two, or more, or all characteristics in the exclusion criteria in the clinical study described in Section 6.4 below. In specific embodiments, a subject to be administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) containing or expressing such a polypeptide(s)), or composition described herein has one, two, or more, or all characteristics in the exclusion criteria in the clinical study described in Section 6.4 below.

[00432] In some embodiments, the human subject to be administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) containing or expressing such a polypeptide(s)), or composition described herein is any individual at increased risk of influenza virus infection or disease resulting from influenza virus infection (e.g., an immunocompromised or immunodeficient individual). In some embodiments, the human subject to be administered an active compound or composition described herein is any individual in close contact with an individual with increased risk of influenza virus infection or disease resulting from influenza virus infection (e.g., immunocompromised or immunosuppressed individuals).

[00433] In some embodiments, the human subject to be administered an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) containing or expressing such a polypeptide(s)), or composition described herein is an individual affected by any condition that increases susceptibility to influenza virus infection or complications or disease resulting from influenza virus infection. In other embodiments, an active compound or composition described herein is administered to a subject in whom an influenza virus infection has the potential to increase complications of another condition that the individual is affected by, or for which they are at risk.

[00434] In certain embodiments, an immunogenic formulation comprising a live virus vector is not given concurrently with other live-virus vaccines. 5.10 MODES OF ADMINISTRATION

5.10.1 Routes of Delivery

[00435] An active compound (e.g., a chimeric hemagglutinin (HA) polypeptide, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) containing, expressing, or both such a polypeptide(s)), or composition described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, transdermal, intravenous, conjunctival and subcutaneous routes. In some embodiments, a composition is formulated for topical

administration, for example, for application to the skin. In a specific embodiment, a composition is formulated for intransal administration. In specific embodiments, the route of administration is nasal, e.g., as part of a nasal spray. In certain embodiments, a composition is formulated for intramuscular administration. In some embodiments, a composition is formulated for subcutaneous administration. In certain embodiments, a composition is not formulated for administration by injection. In specific embodiments for live virus vaccines, the vaccine is formulated for administration by a route other than injection.

[00436] In a specific embodiment, a subunit vaccine is administered intramuscularly. In another embodiment, a live influenza virus vaccine is administered intranasally. In another embodiment, an inactivated influenza virus vaccine (e.g., an inactivated whole virus vaccine, a split influenza virus vaccine, or subunit vaccine) is administered intramuscularly. In another embodiment, a split virus vaccine is administered intramuscularly. In another embodiment, a virus-like particle or composition thereof is administered intramuscularly.

5.10.2 Dosage and Frequency of Administration

[00437] The amount of an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein, a nucleic acid sequence encoding such a polypeptide(s), or a vector (e.g., a viral vector) either containing, expressing, or both such a polypeptide(s)) or composition which will be effective in the treatment and/or prevention of an influenza virus infection or an influenza virus disease will depend on the nature of the disease, and can be determined by standard clinical techniques.

[00438] The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the infection or disease caused by it, and should be decided according to the judgment of the practitioner and each subject' s circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the patient (including age, body weight, health), whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.

[00439] In certain embodiments, the dose of a viral vector (e.g., an influenza virus) described herein may be 10⁴ plaque forming units (PFU) to 10⁸ PFU. In certain embodiments, the dose of a chimeric hemagglutinin (HA) polypeptide described herein (e.g., as provided in split virus vaccines and subunit vaccines) may range from about 1 μg to 150 μg. In certain embodiments, the dose of a chimeric hemagglutinin (HA) polypeptide described herein (e.g., as provided in split virus vaccines and subunit vaccines) is 15 μg. In certain embodiments, the dose for VLPs may range from about 1 μg to about 150 μg of a chimeric HA polypeptide. In certain

embodiments, the dose for VLPs is 15 μg of a chimeric HA polypeptide. In some embodiments, an inactivated vaccine is formulated such that it may contain about 1 μg to about 150 μg of a chimeric hemagglutinin (HA) polypeptide described herein. In some embodiments, an inactivated vaccine is formulated such that it contain 15 μg of a chimeric hemagglutinin (HA) polypeptide described herein.

[00440] In certain embodiments, the dose for administration to a human subject is a dose described in Section 6 below.

[00441] Exemplary doses for nucleic acids encoding a chimeric hemagglutinin (HA) polypeptide described herein range from about 10 ng to 1 g nucleic acid, e.g., DNA, cDNA, or RNA per patient.

[00442] In certain embodiments, a composition described herein (e.g., an immunogenic composition) comprises a human dose volume of 0.05 ml to 1 ml of a liposomal adjuvant described in Section 5.8.5 above, and/or Section 6 below. In certain embodiments, the composition comprises a human dose volume of 0.4 ml to 0.6 ml of the liposomal adjuvant. In certain embodiments, the liposomal adjuvant comprises 1-100 μg of a TLR4 agonist per human dose. In certain embodiments, the liposomal adjuvant comprises 40-60 μg of a TLR4 agonist per human dose. In certain embodiments, the liposomal adjuvant comprises 20-30 μg of a TLR4 agonist per human dose. In certain embodiments, the liposomal adjuvant comprises 1-100 μg of the saponin per human dose. In certain embodiments, the liposomal adjuvant comprises 20-30 μg of the TLR4 agonist and 20-30 μg of saponin per human dose. In certain embodiments, the liposomal adjuvant comprises 40-60 μg of the TLR4 agonist and 40-60 μg of saponin per human dose.

5.11 BIOLOGICAL ASSAYS

[00443] Also provided herein are biological assays that may be used to characterize a chimeric HA, nucleic acid sequence encoding such chimeric HA, and viruses containing, expressing, or both such chimeric HA. See, also, Section 6 below. In a specific embodiment, an assay described in Section 6 below is used to characterize a chimeric HA, a nucleic acid sequence encoding such chimeric HA, or virus containing, expressing, or both such chimeric HA. In another specific embodiment, the immunogenicity or effectiveness of an immunogenic composition described herein is assessed using one, two, or more assays described in Section 6 below.

5.11.1 Assays For Testing Activity Of Chimeric Influenza Virus Hemagglutinin

Polypeptides

[00444] Assays for testing the expression of a chimeric hemagglutinin (HA) polypeptide in a vector disclosed herein may be conducted using any assay known in the art. For example, an assay for incorporation into a viral vector comprises growing the virus as described in this Section, Section 5.4 above or Section 5.5 above, purifying the viral particles by centrifugation through a sucrose cushion, and subsequent analysis for chimeric hemagglutinin (HA)

polypeptide expression by an immunoassay, such as Western blotting, using methods well known in the art. Methods for determining whether a hemagglutinin polypeptide is chimeric are known to those of skill in the art {see, e.g., the Examples 3 and 4 of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety).

[00445] In one embodiment, a chimeric hemagglutinin (HA) polypeptide disclosed herein is assayed for proper folding and functionality by testing its ability to bind specifically to a neutralizing antibody directed to an influenza virus hemagglutinin polypeptide, such as the stalk region of the chimeric HA polypeptide, using any assay for antibody-antigen interaction known in the art. Neutralizing antibodies for use in such assays include, for example, the neutralizing antibodies described in Ekiert et al, 2009, Science Express, 26 February 2009; Kashyap et al, 2008, Proc Natl Acad Sci USA 105: 5986-5991; Sui et al. 2009, Nature Structural and

Molecular Biology, 16:265-273; Wang et al, 2010, P LOS Pathogens 6(2): 1-9; U.S. Patent Nos. 5,589,174, 5,631,350, 6,337,070, and 6,720,409; International Application No.

PCT/US2007/068983 published as International Publication No. WO 2007/134237; International Application No. PCT/US2008/075998 published as International Publication No. WO

2009/036157; International Application No. PCT/EP2007/059356 published as International Publication No. WO 2008/028946; and International Application No. PCT/US2008/085876 published as International Publication No. WO 2009/079259. These antibodies include CR6261, CR6325, CR6329, CR6307, CR6323, 2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, H98, C179 (FERM BP-4517), ADC (FERM BP-4516), among others.

[00446] In another embodiment, a chimeric hemagglutinin (HA) polypeptide disclosed herein is assayed for proper folding by determination of the structure or conformation of the chimeric hemagglutinin (HA) polypeptide using any method known in the art such as, e.g., NMR, X-ray crystallographic methods, or secondary structure prediction methods, e.g., circular dichroism.

[00447] In another embodiment, a chimeric HA disclosed herein is assayed for retention of one, two, or more, or all of the functions of a wild-type influenza HA. Nonlimiting examples of functions of a wild-type influenza HA include fusogenic activity, receptor binding activity, budding, and particle formation. In a specific embodiment, a chimeric HA disclosed herein is assayed for fusogenic activity. Assays known to one skilled in the art can be utilized the assess the fusogenic activity of a chimeric influenza hemagglutinin (HA) polypeptide described herein, such as, for example, immunofluorescence assays and pseudotyped virus-like-particle assays. In certain embodiments, the activity of a chimeric HA polypeptide described herein is assessed in one or more of the following assays: hemagglutination assay(s), fusion assay(s) or budding assay(s).

5.11.2 Assays For Testing Activity Of Antibodies Generated Using Chimeric Influenza

Virus Hemagglutinin Polypeptides

[00448] Antibodies generated using a chimeric HA polypeptide may be characterized in a variety of ways known to one of skill in the art {e.g. ELISA, Surface Plasmon resonance display (BIAcore), Western blot, immunofluorescence, immunostaining and/or microneutralization assays. In some embodiments, antibodies are assayed for the ability to specifically bind to a chimeric hemagglutinin (HA) polypeptide, or a vector comprising said polypeptide. Such an assay may be performed in solution (e.g., Houghten, 1992, Bio/Techniques 13 :412 421), on beads (Lam, 1991, Nature 354:82 84), on chips (Fodor, 1993, Nature 364:555 556), on bacteria (U.S. Patent No. 5,223,409), on spores (U.S. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al, 1992, Proc. Natl. Acad. Sci. USA 89: 1865 1869) or on phage (Scott and Smith, 1990, Science 249:386 390; Cwirla et a/., 1990, Proc. Natl. Acad. Sci. USA 87:6378 6382; and Felici, 1991, J. Mol. Biol. 222:301 310) (each of these references is incorporated herein in its entirety by reference).

[00449] Specific binding of an antibody to a chimeric hemagglutinin (HA) polypeptide or a domain thereof and cross-reactivity with other antigens can be assessed by any method known in the art. Immunoassays which can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety).

[00450] The binding affinity of an antibody to a chimeric hemagglutinin (HA) polypeptide or a domain thereof and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody for a chimeric hemagglutinin (HA) polypeptide and the binding off-rates can be determined from the data by Scatchard plot analysis.

Competition with a second antibody can also be determined using radioimmunoassays. In this case, a chimeric hemagglutinin (HA) polypeptide is incubated with the test antibody conjugated to a labeled compound (e.g., ³H or ¹²⁵I) in the presence of increasing amounts of an unlabeled second antibody. [00451] In a specific embodiment, the binding affinity of an antibody to a chimeric HA polypeptide or a domain thereof is determined using an assay described in Nachbagauer et al, mBio. 2016 Jan-Feb; 7(1): e01996-15.

[00452] In certain embodiments, antibody binding affinity and rate constants are measured using the KinExA 3000 System (Sapidyne Instruments, Boise, ID). In some embodiments, surface plasmon resonance {e.g., BIAcore kinetic) analysis is used to determine the binding on and off rates of the antibodies to an influenza virus hemagglutinin polypeptide. In specific embodiments, an assay described in Tan et al, PLoS Pathog. 2016 Apr; 12(4): el005578 is used to determine the binding on and off rates of antibodies to a chimeric HA polypeptide.

[00453] The neutralizing activity of an antibody can be determined utilizing any assay known to one skilled in the art. Antibodies described herein can be assayed for their ability to inhibit the binding of an influenza virus, or any other composition comprising a chimeric hemagglutinin (HA) polypeptide, to its host cell receptor (i.e., sialic acid) using techniques known to those of skill in the art. In a specific embodiment, an assay described in one of the following articles is used to determine the neutralizing activity of an antibody Tan et al, PLoS Pathog. 2016 Apr; 12(4): el005578; Pica et al, Proc Natl Acad Sci U S A. 2012 Feb 14; 109(7): 2573-2578; and Nachbagauer et al, mBio. 2016 Jan-Feb; 7(1): e01996-15.

[00454] In other embodiments, an antibody suitable for use in the methods described herein does not inhibit influenza virus receptor binding, yet is still found to be neutralizing in an assay described herein. In some embodiments, an antibody suitable for use in accordance with the methods described herein reduces or inhibits virus-host membrane fusion in an assay known in the art or described herein.

[00455] In one embodiment, virus-host membrane fusion is assayed in an in vitro assay using an influenza virus containing a reporter and a host cell capable of being infected with the virus. An antibody inhibits fusion if reporter activity is inhibited or reduced compared to a negative control {e.g., reporter activity in the presence of a control antibody or in the absence of antibody). In a specific embodiment, a reporter assay described in Heaton et al, J Virol. 2013 Aug;87(15):8272-81 is used.

5.11.3 Cytotoxicity Assays

[00456] Many assays well-known in the art can be used to assess viability of cells (infected or uninfected) or cell lines following exposure to an active compound or a composition thereof and, thus, determine the cytotoxicity of the compound or composition. For example, cell proliferation can be assayed by measuring Bromodeoxyuridine (BrdU) incorporation {See, e.g., Hoshino et al, 1986, Int. J. Cancer 38, 369; Campana et al, 1988, J. Immunol. Meth. 107:79), (3H) thymidine incorporation {See, e.g., Chen, J., 1996, Oncogene 13 : 1395-403; Jeoung, J., 1995, J. Biol. Chem. 270: 18367 73), by direct cell count, or by detecting changes in transcription, translation or activity of known genes such as proto-oncogenes {e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, Dl, D2, D3, E, etc). The levels of such protein and mRNA and activity can be determined by any method well known in the art. For example, protein can be quantitated by known immunodiagnostic methods such as ELISA, Western blotting or immunoprecipitation using antibodies, including commercially available antibodies. mRNA can be quantitated using methods that are well known and routine in the art, for example, using northern analysis, RNase protection, or polymerase chain reaction in connection with reverse transcription. Cell viability can be assessed by using trypan-blue staining or other cell death or viability markers known in the art. In a specific embodiment, the level of cellular ATP is measured to determined cell viability.

[00457] In specific embodiments, cell viability is measured in three-day and seven-day periods using an assay standard in the art, such as the CellTiter-Glo Assay Kit (Promega) which measures levels of intracellular ATP. A reduction in cellular ATP is indicative of a cytotoxic effect. In another specific embodiment, cell viability can be measured in the neutral red uptake assay. In other embodiments, visual observation for morphological changes may include enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes are given a designation of T (100% toxic), PVH (partially toxic-very heavy-80%), PH (partially toxic-heavy-60%), P (partially toxic-40%), Ps (partially toxic-slight-20%), or 0 (no toxicity-0%), conforming to the degree of cytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration (ICso) is determined by regression analysis of these data.

[00458] In a specific embodiment, the cells used in the cytotoxicity assay are animal cells, including primary cells and cell lines. In some embodiments, the cells are human cells. In certain embodiments, cytotoxicity is assessed in one or more of the following cell lines: U937, a human monocyte cell line; primary peripheral blood mononuclear cells (PBMC); Huh7, a human hepatoblastoma cell line; 293 T, a human embryonic kidney cell line; and THP-1, monocytic cells. In certain embodiments, cytotoxicity is assessed in one or more of the following cell lines: MDCK, MEF, Huh 7.5, Detroit, or human tracheobronchial epithelial (HTBE) cells.

[00459] Active compounds or compositions thereof can be tested for in vivo toxicity in animal models. For example, animal models, described herein and/or others known in the art, used to test the activities of active compounds can also be used to determine the in vivo toxicity of these compounds. For example, animals are administered a range of concentrations of active compounds. Subsequently, the animals are monitored over time for lethality, weight loss or failure to gain weight, and/or levels of serum markers that may be indicative of tissue damage {e.g., creatine phosphokinase level as an indicator of general tissue damage, level of glutamic oxalic acid transaminase or pyruvic acid transaminase as indicators for possible liver damage). These in vivo assays may also be adapted to test the toxicity of various administration mode and/or regimen in addition to dosages.

[00460] The toxicity and/or efficacy of an active compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. An active compound that exhibits large therapeutic indices is preferred. While an active compound that exhibits toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[00461] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of an active compound for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any active compound used in a method described herein, the effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 {i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high-performance liquid chromatography. Additional information concerning dosage determination is provided herein.

[00462] Further, any assays known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of the active compounds and compositions described herein, for example, by measuring viral infection or a condition or symptoms associated therewith.

[00463] The cytotoxicity assays described herein and known to those skilled in the art are particularly useful for live attenuated influenza viruses.

5.11.4 In vivo Antiviral Activity

[00464] Active compounds and compositions thereof are preferably assayed in vivo for the desired therapeutic or prophylactic activity prior to use in humans {see, e.g., Section 6 below). For example, in vivo assays can be used to determine whether it is preferable to administer an active compound or composition thereof and/or another therapy. For example, to assess the use of an active compound or composition thereof to prevent an influenza virus disease, the composition can be administered before the animal is infected with influenza virus.

Alternatively, or in addition, an active compound or composition thereof can be administered to the animal at the same time that the animal is infected with influenza virus. To assess the use of an active compound or composition thereof to treat an influenza virus infection or disease associated therewith, the compound or composition may be administered after infecting the animal with influenza virus. In a specific embodiment, an active compound or composition thereof is administered to the animal more than one time.

[00465] Active compounds and compositions thereof can be tested for antiviral activity in animal model systems including, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, ferrets, goats, sheep, dogs, rabbits, guinea pigs, etc. In a specific embodiment, active compounds and compositions thereof are tested in a mouse model system. Such model systems are widely used and well-known to the skilled artisan. In a specific embodiment, active compounds and compositions thereof are tested in a mouse model system. Non-limiting examples of animal models for influenza virus are provided in this Section.

[00466] In general, animals are infected with influenza virus and concurrently or subsequently treated with an active compound or composition thereof, or placebo. Alternatively, animals are treated with an active compound or composition thereof or placebo and subsequently infected with influenza virus. Samples obtained from these animals (e.g., serum, urine, sputum, semen, saliva, plasma, or tissue sample) can be tested for viral replication via well known methods in the art, e.g., those that measure altered viral titers (as determined, e.g., by plaque formation), the production of viral proteins (as determined, e.g., by Western blot, ELISA, or flow cytometry analysis) or the production of viral nucleic acids (as determined, e.g., by RT-PCR or northern blot analysis). For quantitation of virus in tissue samples, tissue samples are homogenized in phosphate-buffered saline (PBS), and dilutions of clarified homogenates are adsorbed for 1 hour at 37°C onto monolayers of cells (e.g., Vero, CEF or MDCK cells). In other assays,

histopathologic evaluations are performed after infection, preferably evaluations of the organ(s) the virus is known to target for infection. Virus immunohistochemistry can be performed using a viral-specific monoclonal antibody.

[00467] The effect of an active compound or composition thereof on the virulence of a virus can also be determined using in vivo assays in which the titer of the virus in an infected subject administered an active compound or composition thereof, the length of survival of an infected subject administered an active compound or composition thereof, the immune response in an infected subject administered an active compound or composition thereof, the number, duration and/or severity of the symptoms in an infected subject administered an active compound or composition thereof, and/or the time period before onset of one or more symptoms in an infected subject administered an active compound or composition thereof, is assessed. Techniques known to one of skill in the art can be used to measure such effects. In certain embodiments, an active compound or composition thereof results in a 0.5 fold, 1 fold, 2 fold, 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater reduction in titer of influenza virus relative to an untreated subject. In some embodiments, an active compound or composition thereof results in a reduction in titer of influenza virus relative to an untreated subject 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 9 logs, or 8 to 9 logs.

[00468] Influenza virus animal models, such as ferret, mouse, guinea pig, squirrel monkey, macaque, and chicken, developed for use to test antiviral agents against influenza virus have been described. See, e.g., Sidwell et al, Antiviral Res., 2000, 48: 1-16; Lowen A.C. et al.

PNAS., 2006, 103 : 9988-92; and McCauley et al, Antiviral Res., 1995, 27: 179-186 and

Rimmelzwann et al, Avian Diseases, 2003, 47:931-933. For mouse models of influenza, non- limiting examples of parameters that can be used to assay antiviral activity of active compounds administered to the influenza-infected mice include pneumonia-associated death, serum al-acid glycoprotein increase, animal weight, lung virus assayed by hemagglutinin, lung virus assayed by plaque assays, and histopathological change in the lung. Statistical analysis is carried out to calculate significance {e.g., a P value of 0.05 or less).

[00469] In other assays, histopathologic evaluations are performed after infection of an animal model subject. Nasal turbinates and trachea may be examined for epithelial changes and subepithelial inflammation. The lungs may be examined for bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles. The alveoli are also evaluated for inflammatory changes. The medium bronchioles are graded on a scale of 0 to 3+ as follows: 0 (normal: lined by medium to tall columnar epithelial cells with ciliated apical borders and basal pseudostratified nuclei; minimal inflammation); 1+ (epithelial layer columnar and even in outline with only slightly increased proliferation; cilia still visible on many cells); 2+ (prominent changes in the epithelial layer ranging from attenuation to marked proliferation; cells disorganized and layer outline irregular at the luminal border); 3+ (epithelial layer markedly disrupted and disorganized with necrotic cells visible in the lumen; some bronchioles attenuated and others in marked reactive proliferation).

[00470] The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal: Lined by medium to tall columnar epithelial cells with ciliated apical border, nuclei basal and pseudostratified. Cytoplasm evident between apical border and nucleus. Occasional small focus with squamous cells); 1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffuse squamous metaplasia of much of the epithelial layer, cilia may be evident focally); 2.5+ (diffuse squamous metaplasia with very few cilia evident). [00471] Virus immunohistochemistry is performed using a viral-specific monoclonal antibody (e.g. NP-, N- or HN-specific monoclonal antibodies). Staining is graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (few infected cells); 1+ (few infected cells, as widely separated individual cells); 1.5+ (few infected cells, as widely separated singles and in small clusters); 2+ (moderate numbers of infected cells, usually affecting clusters of adjacent cells in portions of the epithelial layer lining bronchioles, or in small sublobular foci in alveoli); 3+ (numerous infected cells, affecting most of the epithelial layer in bronchioles, or widespread in large sublobular foci in alveoli).

[00472] In one example, the ability to induce lung lesions and cause infection in an animal model of virus infection is compared using wild-type virus and mock virus. Lung lesions can be assessed as a percentage of lung lobes that are healthy by visual inspection. Animals are euthanized 5 days p.i. by intravenous administration of pentobarbital, and their lungs are removed in toto. The percentage of the surface of each pulmonary lobe that is affected by macroscopic lesions is estimated visually. The percentages are averaged to obtain a mean value for the 7 pulmonary lobes of each animal. In other assays, nasal swabs can be tested to determine virus burden or titer. Nasal swabs can be taken during necropsy to determine viral burden post-infection.

[00473] In one embodiment, virus is quantified in tissue samples. For example, tissue samples are homogenized in phosphate-buffered saline (PBS), and dilutions of clarified homogenates adsorbed for 1 h at 37°C onto monolayers of cells (e.g., MDCK cells). Infected monolayers are then overlaid with a solution of minimal essential medium containing 0.1% bovine serum albumin (BSA), 0.01% DEAE-dextran, 0.1% NaHCCb, and 1% agar. Plates are incubated 2 to 3 days until plaques could be visualized. Tissue culture infectious dose (TCID) assays to titrate virus from PR8-infected samples are carried out as follows. Confluent monolayers of cells (e.g., MDCK cells) in 96-well plates are incubated with log dilutions of clarified tissue homogenates in media. Two to three days after inoculation, 0.05-ml aliquots from each well are assessed for viral growth by hemagglutination assay (HA assay). In a specific embodiment, an animal study such as described in Section 6 below is conducted to assess the potential prophylactic and/or therapeutic utility of an active compound or composition described herein, or an immunization regimen described herein. 5.11.4.1 Assays in Humans

[00474] In a specific embodiment, an active compound or composition described herein or an immunization regimen described herein is assessed in a clinical study, such as described in Example 4 (Section 6.4 below) below.

[00475] In one embodiment, an active compound or composition thereof that modulates replication of an influenza virus are assessed in infected human subjects. In accordance with this embodiment, an active compound or composition thereof is administered to the human subject, and the effect of the active compound or composition on viral replication is determined by, e.g., analyzing the level of the virus or viral nucleic acids in a biological sample (e.g., serum or plasma). An active compound or composition thereof that alters virus replication can be identified by comparing the level of virus replication in a subject or group of subjects treated with a control to that in a subject or group of subjects treated with an active compound or composition thereof. Alternatively, alterations in viral replication can be identified by comparing the level of the virus replication in a subject or group of subjects before and after the administration of an active compound or composition thereof. Techniques known to those of skill in the art can be used to obtain the biological sample and analyze the mRNA or protein expression.

[00476] In another embodiment, the effect of an active compound or composition thereof on the severity of one or more symptoms associated with an influenza virus infection/disease are assessed in an infected subject. In accordance with this embodiment, an active compound or composition thereof or a control is administered to a human subject suffering from influenza virus infection and the effect of the active compound or composition on one or more symptoms of the virus infection is determined. An active compound or composition thereof that reduces one or more symptoms can be identified by comparing the subjects treated with a control to the subjects treated with the active compound or composition. In a specific embodiment, administration of an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein) or composition thereof results in a decrease in hospitalization of a human or population of humans caused by influenza virus disease or infection. In another specific embodiment, administration of an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein) or composition thereof results in a reduced need for

respiratory /breathing assistance in a human or population of humans with an influenza virus disease or infection. In another specific embodiment, administration of an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein) or composition thereof results in a reduced length of illness of a human or population of humans with an influenza virus disease or infection. In another specific embodiment, administration of an active compound (e.g., a chimeric hemagglutinin (HA) polypeptide described herein) or composition thereof results in improvement (e.g., an increase) in lung volume as assessed by, e.g., whole body or lung plethysmography. In another embodiment, an active compound or composition thereof is administered to a healthy human subject and monitored for efficacy as a vaccine (e.g., the subject is monitored for the onset of symptoms of influenza virus infection; the ability of influenza virus to infect the subject; and/or a reduction in/absence of one or more symptoms associated with influenza virus infection). Techniques known to physicians familiar with infectious diseases can be used to determine whether an active compound or composition thereof reduces one or more symptoms associated with the influenza virus disease. See, e.g., Section 6 below.

5.12 ASSESSMENT OF ANTIBODIES IN A SUBJECT

[00477] In another aspect, a chimeric influenza virus hemagglutinin polypeptide described herein, or virus either expressing, containing, or both a chimeric influenza virus hemagglutinin polypeptide described herein, can be used to assess the antibody response of a subject (e.g., a naive subject or an immunized/vaccinated subject) or a population of subjects to an influenza virus hemagglutinin polypeptide (e.g., a chimeric influenza virus hemagglutinin polypeptide (see, e.g., Example 8 of International Publication No. WO 2013/043729, which is incorporated herein by reference in its entirety). In specific embodiments, a chimeric influenza virus hemagglutinin polypeptide or a virus either, containing, expressing, or both, a chimeric influenza virus hemagglutinin polypeptide can be used to assess the presence of stem-specific antibodies in the subject or population of subjects.

[00478] In a specific embodiment, the antibody response of a subject or a population of subjects that has been an immunized/vaccinated with a chimeric influenza virus hemagglutinin polypeptide, or a virus expressing and/or containing a chimeric influenza virus hemagglutinin polypeptide, is assessed to identify the types of stalk-specific antibodies in the subject or population of subjects (see, e.g., Section 6 below). Such an assessment may allow for the identification surrogate markers/endpoints important in determining the clinical response to administration of a, chimeric influenza virus hemagglutinin polypeptide) described herein, or a virus expressing and/or a chimeric influenza virus hemagglutinin polypeptide described herein. In such an approach, a biological sample, e.g., blood, from the subject or population of subjects may be isolated and tested directly for the presence of antibodies, or may be processed (e.g., to obtain sera) and subsequently tested for the presence of antibodies.

[00479] In another specific embodiment, the antibody profile of a naive subject (i.e., a subject that has not been immunized/vaccinated with a chimeric influenza virus hemagglutinin polypeptide described herein, or a virus containing and/or expressing a chimeric influenza virus hemagglutinin polypeptide or a population of naive subjects is assessed to determine whether said subject or population of subjects possesses globular head-specific and/or stem specific antibodies against various influenza virus strains or subtypes. Such an assessment may allow for the generation of a chimeric influenza virus hemagglutinin polypeptide), or viruses containing and/or expressing a chimeric influenza virus hemagglutinin polypeptide, that are suitable for administration to said subject or population of subjects, e.g., a chimeric influenza virus hemagglutinin polypeptide, comprising a head domain to which said subject or population of subjects is naive (does not have antibodies against). Such an assessment may determine an immunization strategy for the patient.

[00480] In another specific embodiment, provided herein is a method of assessing/detecting the presence of antibodies in a subject that are specific for a stem domain of a particular influenza virus strain or subtype comprising contacting in vitro a biological sample (e.g., blood, sera) from said subject with a chimeric influenza virus hemagglutinin polypeptide described herein, wherein said chimeric influenza virus hemagglutinin polypeptide comprises a stem domain from the strain or subtype of interest. See Examples 6-8 of International Publication No. WO 2013/043729 and U.S. Patent Application Publication No. 2015/0132330, each of which is incorporated herein by reference in its entirety, for methods for assessing/detecting the presence of antibodies specific for a stem domain of a particular influenza virus strain or subtype. In another specific embodiment, provided herein is a method of assessing/detecting the presence of antibodies in a subject that are specific for a stem domain of a particular influenza virus strain or subtype comprising contacting in vitro a biological sample (e.g., blood, sera) from said subject with a virus expressing and/or containing a chimeric influenza virus hemagglutinin polypeptide described herein, wherein said chimeric influenza virus hemagglutinin polypeptide comprises a stem domain from the strain or subtype of interest.

[00481] In specific embodiment, the antibody response of a subject administered an active compound or composition described herein may be assessed in a passive transfer assay, such as described in Example 5 (Section 6.5 below), below.

5.13 KITS

[00482] Provided herein is a pharmaceutical pack or kit for immunizing against an influenza virus in a subject comprising one or more containers filled with one or more of the ingredients of the pharmaceutical/immunogenic compositions described herein, such as one or more active compounds provided herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of

pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00483] The kits encompassed herein can be used in accordance with the methods described herein. In one embodiment, a kit comprises an active compound described herein, (e.g., one or more chimeric influenza virus hemagglutinin polypeptides described herein (such as described in Section 5.1 above or Section 6 below)), in one or more containers. In another embodiment, a kit comprises one or more immunogenic compositions described herein in one or more containers. In certain embodiments, a kit comprises a vaccine described herein, e.g., a split virus vaccine, a subunit vaccine, an inactivated influenza virus vaccine, or a live influenza virus vaccine, wherein said vaccine comprises one or more one or more chimeric influenza virus hemagglutinin polypeptides described herein and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). In certain embodiments, a kit comprises a vaccine described herein, e.g., a split virus vaccine, a subunit vaccine, an inactivated influenza virus vaccine, or a live influenza virus vaccine, wherein said vaccine comprises one or more chimeric influenza virus hemagglutinin polypeptides. In a specific embodiment, provided herein are kits comprising a chimeric influenza virus hemagglutinin polypeptide described herein and instructions for using the chimeric influenza virus hemagglutinin polypeptide described herein to assess the antibodies present in a subject. [00484] In certain embodiments, a kit described herein comprises: (a) a first container comprising an immunogenic composition described herein, wherein the immunogenic composition comprises a chimeric influenza virus hemagglutinin (HA) polypeptide described herein (e.g., described in Section 5.1 above or Section 6 below); and (b) a second container comprising a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). In specific embodiments, the immunogenic composition is an inactivated whole virus vaccine. In specific embodiments, the immunogenic composition is a split virus vaccine. In specific embodiments, the immunogenic composition is a subunit vaccine.

[00485] In a certain embodiment, a kit described herein comprises (a) a first container comprising a first immunogenic composition comprising 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 second container comprising a second immunogenic composition comprising an inactivated influenza virus and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below), wherein the inactivated influenza virus comprises 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 from the second HA globular head domain. In a specific embodiment, the kit described herein comprises (a) a first container comprising a first immunogenic composition comprising a live attenuated influenza virus engineered to express a first chimeric hemagglutinin (HA) comprising an influenza virus hemagglutinin globular head domain from an influenza A virus of subtype H5, H8, HI 1 or H12 virus and a stem domain from a HI virus; and (b) a second container comprising a second immunogenic composition comprising an inactivated influenza virus and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below), wherein the inactivated influenza virus comprises a second chimeric HA, wherein the second chimeric HA comprises an influenza virus hemagglutinin globular head domain from an influenza A virus of subtype H5, H8, HI 1 or H12 virus and a stem domain from a HI virus, and wherein the first HA globular head domain is different from the second HA globular head domain. [00486] In certain embodiments, a kit described herein comprises (a) a first immunogenic composition, wherein the first immunogenic composition comprises a first inactivated influenza virus 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, wherein the first HA globular head domain is heterologous to the HA stem domain; and (b) a second container comprising a second immunogenic composition, wherein the second immunogenic composition comprises a second inactivated influenza virus and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below), wherein the second inactivated influenza virus comprises 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. For example, the first and second HA globular head domains may be from different subtypes or species. The first inactivated influenza virus may be a whole virus or a split virus. Similarly, the second inactivated influenza virus may be a whole virus or a split virus. The first immunogenic composition may also comprise a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). In certain embodiments, the kit further comprises a third container comprising a third

immunogenic composition, wherein the third immunogenic composition comprises a third inactivated influenza virus, wherein the third inactivated influenza virus comprises a third chimeric HA, wherein the third chimeric HA comprises a third influenza virus HA globular head domain and the HA stem domain, wherein the third globular head domain is heterologous to the HA stem domain, and wherein the third HA globular head domain is different from the first and the second HA globular head domains. The third immunogenic composition may comprise a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). The first, second and third chimeric HA may be ones described in Section 5.1 above or Section 6 below).

[00487] In certain embodiments, a kit described herein comprises (a) a first container comprising a first immunogenic composition, wherein the first immunogenic composition comprises 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, and wherein the HA globular head domain is heterologous to the HA stem domain; and (b) a second container comprising a second immunogenic composition, wherein the second immunogenic composition comprises a second chimeric HA and a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below), 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. The first and second immunogenic compositions may both be subunit vaccines. In a specific embodiment, the first immunogenic composition comprises a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). In certain embodiments, the kit further comprises a third container comprising a third immunogenic composition comprising a third chimeric HA, wherein the third chimeric HA comprises a third influenza virus HA globular head domain and the HA stem domain, wherein the third globular head domain is heterologous to the HA stem domain, and wherein the third HA globular head domain is different from the first and the second HA globular head domains. The third immunogenic composition may comprise a liposomal adjuvant described herein (e.g., in Section 5.8.5 above or Section 6 below). The first, second and third chimeric HA may be ones described in Section 5.1 above or Section 6 below).

6. EXAMPLES

6.1 EXAMPLE 1: CELLULAR IMMUNITY TESTING FOR VACCINATION

WITH ADJUVANTED MONOVALENT CHIMERIC INFLUENZA VIRUS HA POLYPEPTIDE (cHA) VACCINES IN MICE

[00488] This example evaluates the cellular immunity induced by vaccination regimens comprising cHA polypeptides adjuvanted with either AS03 or AS01.

6.1.1 Materials and Methods

(a) Chimeric influenza virus HA polypeptides

[00489] The cH8/l chimeric influenza virus HA polypeptide utilized in this example contains the globular head of domain of A/mallard/Sweden/24/02 (H8N4) HA, and the stalk domain of A/California/04/09 (HlNl) HA. The globular head domain contains the amino acids intervening A_p and A_q and the stalk domain contains amino acids HAlN-term through A_p and A_q through [00490] The cH5/l chimeric influenza virus HA polypeptide utilized in this example contains the globular head domain of A/Vietnam/1203/04 (H5N1) HA and the stalk domain of

A/California/04/09 (HlNl) HA. The globular head domain contains the amino acids intervening Ap and A_q and the stalk domain contains amino acids HAlN-term through A_p and A_q through

[00491] The cH6/l chimeric influenza virus HA polypeptide utilized in this example contains the globular head domain of A/mallard/Sweden/81/02 (H6N1) HA and the stalk domain of A/California/4/2009 (HlNl) HA. The head domain contains the amino acids intervening A_p and A_q and the stalk domain contains amino acids HAlN-term through A_p and A_q through HAlc-term. The stalk domain of the cH6/l chimeric influenza virus HA polypeptide also contains an E47G mutation (HA2 numbering) (see, e.g., Hong et al., 2013, J. Virol., 87(22): 12471-12480 for a description of the E47G mutation).

(b) Influenza viruses, mice, vaccinations and infections

[00492] A/Netherlands/602/2009 (a 2009 pandemic HlNl virus ("HlNlpdm09")) was grown in 8-10 day old embryonated chicken eggs (Charles River Laboratories) at 37°C for 48 hours. Infected eggs were chilled to 4°C for three hours, harvested and sterile clarified by 0.22 μπι filtration (EMD Millipore). Viruses were titered by plaque assay on MDCK cells in the presence of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin.

[00493] For the live-attenuated vaccine ("LAIV") component in this example, cH8/l LAIV was utilized. As used in this example, cH8/l LAIV is a cold-adapted A/Leningrad/134/17/57 master donor virus expressing the cH8/l chimeric influenza virus HA polypeptide and the Nl NA from the 2009 HlNl pandemic virus A/California/04/2009. cH8/l LAIV was rescued and grown at 33°C for 72 hours. Infected eggs were chilled to 4°C for three hours, harvested and sterile clarified by 0.22 μπι filtration (EMD Millipore). Viruses were titered by plaque assay on MDCK cells in the presence of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin.

[00494] Inactivated influenza vaccines ("IIV") for CH8/1N1 and CH5/1N1 were produced in a standard process for split virus vaccines by GlaxoSmithKline and provided as lyophilized preparations. Briefly, recombinant influenza viruses expressing cH8/l or cH5/l in an influenza A/Puerto Rico/8/1934 virus backbone were generated and split with detergent to produce "CH8/1N1" and "CH5/1N1" IIVs, respectively. At the time of vaccination, the lyophilized product was reconstituted either with phosphate buffer diluent for the non-adjuvanted group or with AS03 or AS01 (specifically, ASO IB) Adjuvant System for the adjuvanted groups. ASO IB is composed of immunostimulants QS-21 (a tnterpene glycoside purified from the bark of Quillaja saponaria) and MPL (3-D monophosphoryl lipid A) with liposomes as vehicles for these immunostimulants. A single dose container of ASO IB (0.5 mL) contains 50 μg of QS-21 and 50 μg of MPL.

[00495] The reconstitution of the split virus vaccine was performed as follows:

[00496] (1) Reconstitution with phosphate buffer diluent: with an empty syringe, 625 μΐ. of the phosphate buffer diluent was removed from the vial and injected through the septum into the vial containing the freeze-dried material. This was mixed gently by shaking until the lyophilized cake was dissolved completely.

[00497] (2) Reconstitution with AS03 : 625 μΐ. of AS03 pre-filled syringe ("PFS") was injected through the septum into the vial containing the freeze-dried material. This was mixed gently by shaking until the lyophilized cake was dissolved completely.

[00498] (3) Reconstitution with AS01 With a graduated syringe, 625 μL· of AS01 was injected into the vial containing the freeze-dried material. This was mixed gently by shaking until the lyophilized cake was dissolved completely.

[00499] The quadrivalent seasonal influenza virus vaccine ("QIV") contains split viruses H1N1 A/Christchurch/16/2010 (33 ug HA/mL; H3N2 A/Hong Kong/4801/2014 (36 ug HA/mL); B/Brisbane/60/2008 (36 ug HA/mL; B/Phuket/3073/2013 (37 ug HA/mL).

[00500] Female C57/BL 6 mice aged 6-8 weeks were obtained from Charles River

Laboratories, Wilmington, MA, and housed under specified pathogen-free conditions with food and water ad libitum. Mice under mild ketamine (57 mg/kg) and xylazine (8.6 mg/kg) anesthesia were challenged by intranasal administration of 50 μΐ virus preparation diluted in phosphate buffered saline ("PBS"). All mouse procedures were approved in advance by the Institutional Animal Care and Use Committee (Protocol #IACUC-2015-01 19) at the Icahn School of

Medicine at Mount in accordance with the Animal Act PL99-158 (as amended) and guidelines stated in the 'Guide for the Care and Use of Laboratory Animals' .

(c) Study Design

[00501] Nine groups of 10 mice received a total of three vaccinations on days 0, 28 and 56 (FIG. 2). All groups, except for a mock control group, received an intramuscular, non- adjuvanted QIV (1.5 μg/strain) prime (Table 20). To test the impact of adjuvants on IIV, three groups were boosted with lyophilized CH8/1N1 IIV (1.5 ug of HA) followed by CH5/1N1 IIV (1.5 μg of HA) reconstituted with either ASOl (50 μΐ), AS03 (50 μΐ) or without adjuvant (50 μΐ of phosphate buffer). To measure the impact of LAIV priming, three groups were vaccinated with 10⁵ PFU of CH8/1N1 LAIV, followed by either AS03-adjuvanted CH5/1N1 IIV (1.5 μg of HA), non-adjuvanted CH5/1N1 IIV (1.5 μg of HA) or a mock infection with allantoic fluid. The impact of an IIV prime was tested with groups that either received AS03-adjuvanted CH5/1N1 IIV (1.5 μg of HA) or a mock infection with allantoic fluid on day 28, followed by vaccination with 10⁵ PFU of CH8/1N1 LAIV on day 56. The mock control group received PBS on day 0, allantoic fluid intranasally on day 28 and AS03 on day 56.

[00502] Table 20. Vaccination groups.

[00503] On day 66 (10 days post final vaccination), five mice per group were euthanized and tissues were collected to measure post-vaccination T cell responses. On day 84, the remaining five mice per group were challenged with 5xLDso (5 times lethal dose 50) of

A/Netherlands/602/2009 (HlNlpdm09 virus). On day 6 post-challenge, tissues were collected to measure T cell responses and viral titers in the lung.

(d) Serological analysis

[00504] Blood was collected by submandibular bleeding on days 28 (28 days post prime), 56 (28 days post boost #1) and 84 (28 days post boost #2). Blood clotting was allowed at 4°C overnight and serum was prepared by double centrifugation (16100xg) before storage at -20°C until further analysis.

[00505] Recombinant cH6/l protein was produced using the baculovirus expression system in insect cells (see, e.g., Krammer et al., 2012, PLoS ONE, 7(8):e43603). Serum samples were pooled for ELISA analysis and tested in technical triplicates.

[00506] High-binding 96-well ELISA plates (Thermo Scientific) were coated with 50 μΐ of antigen diluted at a concentration of 2 μg/ml in coating buffer (KPL). The plates were incubated for 12-18 hours at 4 °C and the coating solution was removed with three washes of PBS-T (PBS, 0.1% Tween-20, pH 7.4). The plates were blocked with 220 μΐ of blocking solution (PBS-T with 3% goat serum (Life Technologies) and 0.5% milk powder) per well for 1 hour at room temperature. Blocking solution was replaced with 100 μΐ of fresh blocking solution per well. For 3 -fold dilutions, additional 40 μΐ of blocking solution were added to the first well of each dilution series. Ten microliters of pre-diluted serum was added to the first well of each dilution series. Fifty microliters was transferred from well to well. Two columns per plate did not contain any serum and were used as blanks. The plates were incubated for 2 hours at room temperature and then washed three times with PBS-T. Horse radish peroxidase-labelled anti- mouse IgG (whole molecule, Sigma) was used as secondary antibody. Fifty microliters of secondary antibody diluted at a concentration of 1 :3000 in blocking solution was added to each well and the plates were incubated for 1 hour at room temperature. The plates were washed four times and developed with 100 μΐ of SigmaFast OPD (Sigma) substrate per well for 10 min. Enzymatic color development was stopped with 50 μΐ of 3 molar hydrochloric acid per well and the plates were read at an absorbance of 490 nm. The average of blank values + three standard deviations was calculated for each plate and plates that exceeded a value of 0.075 were repeated. The results are reported as area under the curve values. Data were analyzed using Microsoft Excel 15 and Graphpad Prism 7.

(e) Lung virus titers

[00507] Mice from each group were euthanized on day 6 after infection and lungs were removed aseptically. Left lung lobes were homogenized in 0.3 ml of PBS. The homogenates were centrifuged (15 min, 16100xg, 4°C) to remove cellular debris and stored in single use aliquots at -80° C. Titers of infectious virus were determined by plaque assay. Briefly, 250 μΐ of tenfold dilutions of lungs homogenized in PBS were used to infect confluent monolayers of MDCK cells. Vims was allowed to attach to MDCK cells for 1 hour at 37°C. Cells were washed once with PBS and overlay ed with oxoid agar (Oxoid Ltd., Basingstoke, Hampshire) prepared using NaHCCb-buffered serum-free 2x MEM/B A containing DEAE Dextran and supplemented with TPCK-treated trypsin. Endpoint virus titers were determined by visualizing virus plaques 2 days after infection by staining with polyclonal serum, horseradish peroxidase- conjugated sheep-derived anti-mouse serum (GE Healthcare UK, Chalfont St Giles, UK) and TrueBlue substrate (KPL-Seracare, Milford, MA).

(f) Immune cell isolation and IFN-gamma enzyme-linked immunospot assay

[00508] Whole lungs and spleens were isolated aseptically on day 10 after the last vaccination (n=5 mice/group) or right lung lobes and spleens were isolated aseptically on day 6 after infection (n=5 mice/group). Lung tissue was cut into 1 mm³ cubes and digested with type IV collagenase (Worthington, Freehold, NJ) for 30 minutes at 37° C. Single cell suspensions of spleens and digested lungs were prepared by forcing tissue over a 70 μιη mesh cell strainer (Corning, Oneonta, NY). Red blood cells were lysed by resuspension of cells in 5 mL of NH₄C1 solution. Lung cells were further enriched by centrifugation of lung cell suspensions (400g for 5min) in 33% Percoll Plus (GE, Rahway, NJ).

[00509] T cell analysis was performed according to the immuno-plate manufacturer's protocol (R&D Systems, Minneapolis, MN). Briefly, 96-well immuno-plates pre-coated with sterile monoclonal anti-IFN-γ antibodies were blocked with culture medium (RPMI1640 supplemented with 10% fetal calf serum, L-glutamine and antibiotics). Approximately 10⁵ cells were plated in 100 μΐ of culture medium supplemented with peptides or whole virus. Exact cell counts were measured using a Gallios flow cytometer (Beckman-Coulter, Indianapolis, IN) operated with Kaluza software. High-pressure liquid chromatography-purified (>95% purity) H2b-restricted epitope-specific peptides (Influenza virus NP-derived ASNENMETM (SEQ ID NO: 100) and Human Papilloma virus E7-derived RAHYNIVTF (SEQ ID NO: 104) irrelevant peptide) were purchased from Anaspec (Fremont, CA). Pools of 15-mer peptides with 11 amino acid overlaps spanning either the influenza nucleoprotein or hemagglutinin protein of A/California/4/2009 (HlNlpdm09) virus were obtained from Miltenyi (Cambridge, MA) and used at the dilution recommended by the manufacturer. A/Netherlands/602/2009 (HlNlpdm09) was used at 10⁴ PFU/well for restimulation. After overnight restimulation, plates were washed with enzyme- linked immunosorbent assay wash buffer and IFN-γ trapped on the plates was detected by a biotinylated polyclonal anti-IFN-γ antiserum. BCIP/NBT chromogen substrate for alkaline phosphatase conjugated to streptavidin resulted in the formation of spots at places where immune cells secreted IFN-γ during peptide-restimulation. The spots were counted using an ImmunoSpot S6 Micro Analyzer (Cellular Technology Ltd (CTL), Shaker Heights, OH) with ImmunoCapture 6.4 software (CTL) and results are expressed as spots/10⁶ cells or spots per organ.

6.1.2 Results

[00510] T cell responses for chimeric HA vaccines were investigated by ELISPOT in lung and spleen after vaccination and challenge to measure T cell responses elicited by cHA IIV vaccinations with or without adjuvant, compared to responses elicited by cHA LAIV

vaccinations. All mice (except for a mock vaccination group) were primed with non-adjuvanted QIV, to prime the animals to influenza viruses. These mice were then vaccinated twice (28 and 56 days) after the prime. Importantly, a dominant MHCI-restricted epitope on NP that was used as an antigen for measuring T cell responses was drifted between the IIV (NP from PR8), LAIV (NP from Leningrad) and the challenge strain (NP from HlNlpdm09) (Table 21).

[00511] Table 21 : NP sequence homology of dominant epitope.

(a) LAIV vaccination elicits high but transient T cell responses measured in the spleen

[00512] Antigen dependent T cell responses were analyzed in the spleen 10 days post final vaccination (FIGS. 3 A-C). Cells were stimulated either with a CD8-restricted dominant NP epitope from PR8 (ASNENMETM (SEQ ID NO: 100), identical to IIV vaccine strains), whole A/Netherlands/602/2009 HlNlpdm09 virus or an irrelevant peptide. Trends between

ASNENMETM (SEQ ID NO: 100) and whole virus-stimulated samples were overall similar for T cell responses measured in spleens. Some unspecific activity of IFN-y-producing cells could be detected at baseline (cells stimulated with irrelevant peptide) (FIG. 3C). ASNE METM (SEQ ID NO: 100)-specific CD8+ T cell levels were highest in the group that received mock- LAIV, suggesting LAIV administration resulted in efficient induction of CD8+ T cell responses (FIG. 3 A). LAIVs have previously shown to potently induce T cells in humans and animal models (see, e.g., Mohn et al., 2015, J Infect Dis 211(1): 1541-1549 and Mueller et al., 2010, J Virol, 84(4): 1847-1855, each of which is incorporated by reference herein in its entirety).

However, the group that received LAIV-mock showed about a 14-fold lower number of IFN-y+ cells per spleen, indicating that ASNENMETM (SEQ ID NO: 100)-specific CD8+ T cell levels induced by LAIV were only transiently induced in the spleen. Vaccination with split inactivated vaccine, prior to LAIV (IIV-LAIV vaccinated group) induced on average slightly lower numbers of ASNENMETM (SEQ ID NO: 100)-specific CD8+ T cells compared to mock-LAIV. This difference was not seen for splenocytes stimulated with whole virus (FIG. 3B), but could suggest that immunity provided by IIV may slightly reduce but not inhibit T cell induction by LAIV. Mice that received LAIV first followed by IIV with AS03 adjuvant showed a similarly low number of CD8+ T cells compared to those that received no IIV boost (FIG. 3 A and FIG. 3B). T cell responses in the spleen were low for groups that only received IIV vaccines, but two mice in the ASOl-adjuvanted group induced moderately high levels of IFN-γ producing CD8+ T cells.

(b) ASOl-adjuvanted vaccination induces influenza virus specific T cells in the lung.

[00513] T cell responses in lungs were quantified in parallel to those measured in spleens. The number of unspecific IFN-y-producing cells at baseline was lower compared to the spleen (FIG. 4C). As observed for splenocytes, the number of ASNENMETM (SEQ ID NO: 100)- specific CD8+ T cells was the highest in the lungs when LAIV was given last (mock-LAIV compared to LAIV-mock) (FIG. 4A). In contrast, when cells from lungs were stimulated with whole virus (FIG. 4B), the number of IFN-y+ cells did not show a difference between groups that were given an LAIV vaccine as the 1st or 2nd boost. Moreover, upon stimulation with whole virus, the number of IFN-y+ cells induced by vaccination with IIV-IIV adjuvanted with AS01 exceeded all other vaccination regimens (FIG. 4B). AS03-adjuvanted IIV-IIV vaccination resulted in slightly higher levels of IFN-y+ cells after whole virus stimulation compared to non- adjuvanted IIV-IIV vaccination, which was not seen during stimulation with ASNENMETM (SEQ ID NO: 100) peptide. This finding was surprising, since induction of T cell responses in the lung by split virus vaccines is generally not described, compared to vaccination with a live virus vaccines, which potently induce T cell responses in the lung.

(c) Mice that received LAIV last showed low T cell responses in the spleen after lethal virus challenge.

[00514] Three weeks post final vaccination, mice were challenged with 5xLD50 of

HlNlpdm09 and T cell responses were analyzed 6 days after challenge. The time point was chosen such that T cell responses observed were more likely to be a recall response rather than due to de novo T cell priming. This was illustrated by the absence of major T cell responses in the spleen and lung after infection of mice that had been mock-vaccinated (mock-mock-mock) (FIG. 5A-FIG. 5E and FIG. 6A-FIG. 6D). The immunodominant H2b -restricted NP epitope (ASNENMETM (SEQ ID NO: 100)) is derived from A/Puerto Rico/8/1934 ("PR8") virus and is therefore conserved in PR8-derived vaccines (IIV), but is drifted in the Leningrad vaccine strain (LAIV), the NP-overlap pool peptide (A/Cal/04), and the challenge strain (A/NL/602) (Table 21). This drift from the challenge strain may explain why the overall T cell response in the spleen towards the ASNENMETM (SEQ ID NO: 100) peptide is lower compared to the one quantified with the NP-overlap pool peptide, since the infection may have boosted T cells that reacted more strongly with the drifted epitope. The dominant Hlb-restricted epitope in the NP- overlap pool peptide (A/Cal/04) matches better to the one in the challenge virus than

ASNENMETM (SEQ ID NO: 100) (Table 21). Moreover, stimulation with the NP-overlap peptide pool resulted in recall responses of splenic T cells (both CD4+ and CD8+) that recognize epitopes other than ASNENMETM (SEQ ID NO: 100) within NP (FIG. 5 A and FIG. 5B).

Although T cell responses were higher before challenge in groups that received LAIV as the last immunization, these groups had the lowest numbers of IFN-Y+ T cells in the spleen after challenge in all stimulation conditions (FIG. 5A-FIG. 5D). In contrast, groups that received LAIV first, followed by mock vaccination or AS03-adjuvanted IIV vaccination were the ones that had high IFN-Y+ cell counts when T cells were stimulated with NP overlap, whole virus, and HA overlap (A/Cal/04) (FIG. 5B-FIG. 5D).

(d) T cell responses in the lung show high background reactivity post challenge.

[00515] For the analysis of pulmonary T cell responses 6 days after challenge, the right lungs of mice were isolated. The ELISPOT results for all stimulation conditions, including stimulation with irrelevant peptide showed similar activation patterns for all groups (FIG. 6A-FIG. 6D). The presence of replicating live virus post challenge most likely caused T cell stimulation in all conditions, which makes it difficult to assess responses to individual antigens. Of note, animals that received adjuvanted IIV vaccines twice and animals that received LAIV vaccines as the last boost before challenge showed lower levels of T cell activation compared to other vaccinated groups. To test if this was associated with a difference in viral replication, viral titers in the lung were measured by plaque assay.

(e) Lower T cell recall responses in vaccinated animals were associated with lower lung viral titers post challenge.

[00516] To measure the viral load in the lung, the left lungs were collected on day 6 post challenge. Interestingly, mice that received LAIV as the last vaccination successfully controlled viral replication, while animals that received LAIV followed by either IIV or mock vaccination had higher viral titers in the lung (FIG. 7A). This observation is consistent with the transient upregulation of T cells in the lung seen in the LAIV groups post vaccination. In addition, this effect could be partially mediated by an upregulation of innate immunity after LAIV vaccination, that wanes after a month post vaccination. Notably, mice that received AS01 -adjuvanted IIV controlled viral replication similarly well as the groups that received LAIV last. The protection in this group was likely conferred by a combination of antibodies and cellular immune responses. Antibody levels against the HI stalk domain were similarly high in groups that received AS01- or AS03-adjuvanted vaccines, but differed more in T cell responses, which could have provided additional protection after challenge (FIG. 7B). The three groups that effectively controlled viral replication had the highest T cell activation post vaccination in the lung (FIG. 4A and FIG. 4B) and showed lower levels of T cell reactivation in the lung post challenge (FIG. 6) compared to groups that moderately controlled viral replication. This indicates that T cell recall responses were elicited by replicating virus in groups that did not fully suppress viral replication after challenge.

[00517] Of note, the timing and order of LAIV - IIV or IIV - LAIV vaccination did not impact on HI stalk antibody levels measured by ELISA. Furthermore, groups that received adjuvanted vaccines reached similar antibody levels and LAIV followed by non-adjuvanted IIV elicited similar levels as non-adjuvanted IIV given twice (FIG. 7B). 6.2 EXAMPLE 2: INFLUENZA VIRUS SUPRA-SEASONAL UNIVERSAL

INFLUENZA VIRUS VACCINE ("SUIV") CHIMERIC INFLUENZA VIRUS HA POLYPEPTIDE IN MICE

[00518] Current seasonal influenza virus vaccines induce antibody responses that are mainly directed against the head domain of the surface glycoprotein HA. These vaccines show good efficacy when they are well-matched with circulating virus strains. However, the surface glycoproteins of influenza viruses undergo constant antigenic changes, which allows the virus to escape pre-existing immunity. This process is referred to as "antigenic drift". Therefore, HA head-based seasonal vaccines have to be re-formulated and re-administered on an annual basis. Protection from influenza viruses is usually correlated with the concentration of specific HA neutralizing antibodies which bind to the globular head domain of the HA molecule which prevents the virus from attaching to host cell receptors or the fusion to the cell membrane.

Without being bound to any particular theory, the supra-seasonal universal influenza virus ("SUIV") chimeric influenza virus HA ("cHA") vaccination approach described herein aims at focusing the immune response towards the more conserved and immuno-subdominant HA stalk domain, rather than the immuno-dominant HA head domain, using cHAs that are serially administered. The cHAs are combinations of "exotic" head domains, mostly from avian influenza virus subtypes to which humans are naive, paired with a conserved HA stalk domain. Due to the conservation of the HA stalk domain within a same group (e.g., influenza A virus Group 1), these antibodies are able to broadly react with a wide spectrum of influenza virus strains and subtypes. It has been demonstrated in mice that cHA-based vaccination regimens induce higher HA stalk domain antibody titers than the seasonal influenza virus vaccine.

[00519] The primary objective of this immunogenicity study was to justify the need for an adjuvant system by comparing the anti-Hl stalk antibody titers (ELISA assay) in adjuvanted groups versus non-adjuvanted groups and QIV control group.

[00520] The secondary objective of this study was to evaluate the impact of the cHA order on the antibody response (e.g., cH8/l followed by cH5/l versus cH5/l followed by cH8/l), and to compare the immune responses induced by AS03A and ASOlB-derived Adjuvant systems with the cHA antigens. 6.2.1 Materials and Methods

(a) Chimeric influenza virus HA polypeptides

[00521] The cH8/l and cH5/l chimeric influenza virus HA polypeptides used in this example are the constructs described in Table 23, infra.

(b) Study design

[00522] Groups of 40 B ALB/c mice ("BALB/cAnNCrl") (divided in two staggered studies with 20 mice per group) were primed (50 μΙ,ΛΙοβε, equivalent to a 1 : 10th of human dose) on day 0 with QIV (Quadrivalent Influenza vaccine containing A/Christchurch/16/10 (HlNl), A/Hong Kong/4801/14 (H3N2), B/Brisbane/60/08, and B/Phuket/3073/13 strains), boosted on day 28 with a CH5/1N1 or CH8/1N1 and then again on day 56 with a CH8/1N1 or CH5/1N1 vaccine non- adjuvanted or adjuvanted with either AS03A or ASOlB-derived as described in Table 22. Control groups were injected twice with QIV or three times with PBS.

[00523] Table 22. Summary of the study design

^XQIV was produced in Dresden under the trade name of Fluarix Tetra®

(c) Investigational products and formulations tested

[00524] The inactivated split CH8/1N1 and CH5/1N1 monovalent antigens {see Table 23) were produced at GSK's Dresden facility following the same production process as the one used for monovalent bulks that are combined into the inactivated split virion seasonal vaccine Fluarix Tetra®. The monovalent SUIV cHA were formulated from the corresponding inactivated split virus antigen and then freeze-dried and supplied as a sterile lyophilized powder.

[00525] The AS03 Adjuvant System is composed of squalene, DL-a-tocopherol, and polysorbate 80.

[00526] The ASOl Adjuvant System is composed of two immunostimulants: 3-0-desacyl-4'- monophosphoryl lipid A (MPL) and the saponin QS-21.

[00527] ASO IB Adjuvant System is composed of immunostimulants QS-21 (a triterpene glycoside purified from the bark of Quillaja saponaria) and MPL® (3-D monophosphoryl lipid A) with liposomes as vehicles for these immunostimulants. In particular, a single dose container of ASO IB (0.5 mL) contains 50 μg of QS-21 and 50 μg of MPL®.

[00528] The ASOl Adjuvant System is pH 6.1 buffered. To conduct the mouse

immunogenicity study, the ASO IB adjuvant was diluted with an equivalent volume of pH 7.2 phosphate buffer diluent. This allowed to obtain an ASOl adjuvant preparation with increased pH (approximately 6.6), and the pH of the SUIV ASOl-adjuvanted corresponding reconstituted vaccine was approximately 6.9. Due to this 1 : 1 dilution, the amounts of the immune-enhancers MPL and QS-21 and the liposomal components l,2-dioleoyl-sn-glycero-3 -phosphatidylcholine ("DOPC") and cholesterol in the administered vaccine will correspond to those of ASO IE (25 μg of QS-21 and 25 μg of MPL®), but since no formulation development studies were performed in advance the designation of "ASOlB-derived" has been adopted.

[00529] Further information on the compounds/ingredients tested in this example is provided in Table 23.

[00530] Table 23. Formulations tested

*Dose of 500 uL equivalent to one human dose.

**Split virus vaccine.

l^rrhe stalk domain contains amino acids HAl_N-term through A_p and A_q through HAlc-tem.

:The head domain contains the amino acids intervening A_p and A_q.

[00531] At the time of the injection, the lyophilized product was reconstituted either with phosphate buffer diluent for the non-adjuvanted group or with AS03 or ASOl for the adjuvanted groups.

[00532] The reconstitution was performed as follows:

[00533] (1) Reconstitution with phosphate buffer diluent: with an empty syringe, 625 μΙ_, of the phosphate buffer diluent was removed from the vial and injected through the septum into the vial containing the freeze-dried material. This was mixed gently by shaking until the lyophilized cake was dissolved completely.

[00534] (2) Reconstitution with AS03 : 625 μΐ, of an AS03 pre-filled syringe (PFS) was injected through the septum into the vial containing the freeze-dried material. This was mixed gently by shaking until the lyophilized cake was dissolved completely.

[00535] (3) Reconstitution with ASOl : with an empty syringe, the whole volume of one vial of phosphate buffer diluent was removed from the vial and injected through the septum into one vial of ASOIB. This was mixed gently. With a graduated syringe, 625 μΙ_, of the diluted ASOl was removed and injected into the vial containing the freeze-dried material. This was mixed gently by shaking until the lyophilized cake was dissolved completely.

(d) Ethical Statement

[00536] Animal husbandry and experiment were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and the GSK Vaccine policy on the care, welfare and treatment of animals. The ethical protocol P004/26/01 (Viral project) Immunogenicity studies in mice (IP, IM && SC immunizations) was approved by the local GSK ethical committee.

(e) Animal model

[00537] The study was conducted in female BLACK/6 mice (BALB/cAnNCrl) that are genetically equivalent to the BALB/c mice used in Nachbagauer et al., 2016, Vaccines 1, Article number: 16015 (2016) doi: 10.1038/npj vaccines. At the beginning of the treatment period, the animals were approximately 6 to 8 weeks old.

(f) Housing and Husbandry

[00538] Upon arrival, the animals were acclimated in polycarbonate cages type IV

(Tecniplast, 1500 cm²) for a period of 9 days. During the acclimation period, each animal was identified by an individual ear tag.

[00539] The animals were randomly allocated to groups, with a maximum of 10 mice per cage type III (Tecniplast, 800 cm²). All animals had free access to food (diet ref. A04-10 maintenance from SAFE) and tap water (filtered with a 0.22 μπι filter).

[00540] Nesting material was provided within the cages with nonstructural enrichment material (Envirodry). Bedding was made of sawdust Litaspen 8/20 (aspen which is heat-treated, dust-free and not treated by chemicals), and bedding change was performed every week.

Certified bedding was purchased from a commercial supplier (Carfil).

[00541] Air supplied in housing room was 100 % fresh air filtered by EPA filter and the ventilation was at least 20 cycles per hour. The animal room conditions were set as follows: temperature: 22°C (+/- 2°C); humidity: 55% (range from 40-65%) and light/dark cycle: 12h/12 h. The pressure, temperature and relative humidity were recorded continuously by probes.

(g) Observations of clinical signs after vaccine administration

[00542] Throughout the study, the animals were observed at day 1, 29 and 57 for clinical signs potentially associated with treatment and a full physical examination of each animal was performed. If necessary, the physical examination was continued on a daily basis until signs were no longer apparent. In addition, all animals were also examined once each week as part of the physical examination. [00543] No abnormal observations were reported following administration of the cHA vaccine candidates and QIV.

(h) Immunological read-outs: Humoral responses - antibody binding by ELISA

[00544] Recombinant cH6/l (head domain from HA of H6N1 virus A/mallard/Sweden/81/02, stalk domain from HA of HlNlpdm09 virus A/California/4/09), H2

(A/mallard/Netherlands/5/00), H3 (A/Hong Kong/4801/2014), H9 (A/chicken/Hong- Kong/G9/1997) and H18 (A/flat-faced bat/Peru/033/10), Nl (A/California/04/09) proteins were used as ELISA substrates. These proteins were produced using the baculovirus expression system in insect cells (see, e.g., Krammer et al., 2012, PLoS One, 7(8):e43603). The serum samples were pooled for ELISA analysis and tested in technical triplicates. In addition, to test statistical significance of the differences in HI stalk antibody titers, individual serum samples from day 84 post prime were tested for reactivity against cH6/l .

[00545] High-binding 96-well ELISA plates (Thermo Scientific) were coated with 50 μΐ of antigen diluted at a concentration of 2 μg/ml in coating buffer (KPL). The plates were incubated for 12-18 hours at 4°C and the coating solution was removed with three washes of PBS-T (PBS, 0.1% Tween-20, pH 7.4). The plates were blocked with 220 μΐ of blocking solution (PBS-T with 3% goat serum (Life Technologies) and 0.5% milk powder) per well for 1 hour at room temperature.

[00546] The blocking solution was replaced with 100 μΐ of fresh blocking solution per well. For 3 -fold dilutions, an additional 40 μΐ of blocking solution was added to the first well of each dilution series. Ten microliters of pre-diluted serum was added to the first well of each dilution series. Fifty microliters was transferred for 3-fold serial dilutions. Two columns per plate did not contain any serum and were used as blanks. The plates were incubated for 2 hours at room temperature and then washed three times with PBS-T. Horse radish peroxidase-labelled anti- mouse IgG (whole molecule, Sigma) was used as secondary antibody. For IgG subtyping ELISAs, Horse radish peroxidase-labelled anti-mouse IgGl (IgGl heavy chain, abeam) and IgG2a (IgG2a heavy chain, abeam) were used. Fifty microliters of secondary antibody diluted at a concentration of 1 :3000 in blocking solution was added to each well and the plates were incubated for lh at room temperature. The plates were washed four times and developed with 100 μΐ of SigmaFast OPD (Sigma) substrate per well for 10 min. Enzymatic color development was stopped with 50 μΐ of 3 molar hydrochloric acid per well and the plates were read at an absorbance of 490 nm. The average of blank values + three standard deviations was calculated for each plate and plates that exceeded a value of 0.075 were repeated. The results are reported as end-point titers. The end-point titer was defined as the last dilution in which the reactivity of a serum sample was still above OD 0.075. Data were analyzed using Microsoft Excel (Version 15.31).

(i) Immunological read-outs: Fc-mediated functionality in vitro assay

[00547] In order to characterize the functional activity of the anti-Hl stalk antibody response in vitro, anti-Hl stalk specific antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated by an ADCC reporter assay.

[00548] MDCK cells were diluted to a concentration of 2xl0⁵ cells/ml in cDMEM and 100 μΐ/well were seeded in white polysterine 96-well assay plates (Costar Corning). Plates were incubated over night at 37°C, 5% CO2. Viruses were diluted to a multiplicity of infection ("MOI") of 2 per 100 μΐ in UltraMDCK media (Lonza). Cells were washed with PBS (Life Technologies) and infected with 2xMOI of chimeric H6/1N5 virus (an influenza virus reassortant having a cH6/l and the A/mallard/Sweden/86/2003 (H12N5) NA and an influenza A/Puerto Rico/8/1934 virus backbone; cH6/l contains the HA globular head domain of

A/mallard/Sweden/81/2002 (H6N1) and the HA stalk domain of A/California/04/2009 (HlNl)) or with 2xMOI of wild-type A/Netherlands/602/09 HlNlpdm09 virus. Plates were incubated overnight at 37°C, 5% CO2. Cells were washed with PBS and 25 μΐ of 2-fold serially diluted mouse sera were added. 1.25xl0⁵ of ADCC Bioeffector mFc'Y' RIV cells (Promega) in 25 μΐ and 25 μΐ of RPMI 1640 media (Life Technologies) were then added to all wells. Plates were incubated for 6 hours at 37°C, 5% CO2 followed by addition of 75 μΐ of Bio-Glo Luciferase substrate (Promega). Luminescence was read after 15 minutes with a Synergy HI hybrid multimode microplate reader (BioTek). Background luminescence (average + 3x standard deviation) of wells without antibody was subtracted and area under the curve calculated in GraphPad Prism. (j) Serum transfer/viral challenge (cH6/lN5, HlNlpdm09)

[00549] The functional activity of the anti-Hl stalk domain antibodies induced by sequential immunization with cHAs was assessed in vivo by passive serum transfer into naive mice, followed by a lethal viral challenge.

[00550] All the mouse procedures were approved in advance by the Institutional Animal Care and Use Committee (Protocol #IACUC-2015-0119) at the Icahn School of Medicine at Mount in accordance with the Animal Act PL99-158 (as amended) and guidelines stated in the 'Guide for the Care and Use of Laboratory Animals'.

[00551] For the passive transfer experiment, 150 μΐ of pooled mice serum (Day 84) from each cHA group, the QIV group, and the PBS control group were transferred intraperitoneally into naive BALB/c mice (6-8 week-old female BALB/c mice, n=10/group, Jackson Laboratories). Two to five hours after the serum transfer, the mice were sedated with an intraperitoneal ketamine/xylazine injection, were retroorbitally bled and were challenged with 10xLD50 (150 plaque forming units ("PFU")) of wild-type HlNlpdm09 (A/Netherlands/602/09) virus, or 10xLD50 (200 PFU) of cH6/lN5 virus. The cH6/lN5 virus has an exotic HA head domain (H6) and a neuraminidase (N5) to which the vaccinated mice are immunologically naive, but the antibodies in the transferred sera will recognize the conserved HI stalk domain of the virus. Weight loss was monitored daily for up to sixteen days. Mice that lost more than 25% of their initial body weight were euthanized for ethical reasons. All pre-challenge blood samples were tested for reactivity to cH6/l antigen by ELISA. Animals that showed no reactivity in the ELISA assay were excluded from further analysis (unsuccessful transfer).

6.2.2 Statistical methods

[00552] The anti-Hl (cH6/l) IgG ELISA response using individual Day 84 serum samples is expressed as end-point antibody titers. The end-point titers were defined as the last dilution in which the reactivity of a serum sample was still above the cutoff of 0.075 OD490.

[00553] A two-way ANOVA model was fitted on loglO anti-cH6/l ELISA titers related to including groups and experiment as fixed effect and using a heterogeneous variance model (identical variances were not assumed between groups). This model was used to estimate geometric means (and their 95% CIs) as well as the geometric mean ratios and their 95% CIs. 6.2.3 Results

(a) Humoral immune responses

(A) HI stalk-IgG antibody responses

[00554] Anti-Hl stalk domain binding IgG antibody responses were measured by ELISA on day 0, day 28 (28 days post- priming), day 54 (28 days post second immunization) and day 84 (28 days post third immunization).

[00555] In QIV primed mice, the first immunization with the cHA formulations elicited detectable anti-Hl stalk antibody responses in all groups which were boosted after the second cHA (heterologous for the HA heads) administration (FIG. 8).

[00556] Higher titers of specific anti-Hl stalk domain IgG antibodies were detected after immunization with the adjuvanted cHA formulations (both AS03A and ASOlB-derived) when compared to the non-adjuvanted formulations (p values <0.001 at day 84) or to the QIV immunization (p value < 0.001 at day 84) (FIG. 8 and FIG. 9). Both adjuvanted formulations, AS03A and ASOlB-derived, induced comparable anti-Hl stalk Ab titers (p>0.05).

[00557] The order of antigen administration (cH8/l followed by cH5/l versus cH5/l followed by cH8/l) had no statistically significant impact on the anti-Hl stalk antibody response on day 28 post last immunization (day 84) (p>0.05).

(B) Cross-reactive anti-HA antibody responses

[00558] HA stalk domains are antigenically conserved within Groups Al, A2 and B. HI, H2, H5 and H6 belong to the HI clade and are highly antigenically conserved. H8 and H9 belong to the H9 clade and HI 8 is a recently identified and divergent influenza virus subtype discovered in the bat (Tong et al., 2013). Cross-reactive stalk specific antibody titers were evaluated by ELISA assasy with Group Al full-length HA (H2, H9 and HI 8) coating antigens, on day 0, day 28 (28 days post- priming), day 54 (28 days post second immunization) and day 84 (28 days post third immunization) (FIG. 10).

[00559] Cross-reactive anti-HA antibody responses were detected against H2, H9, and HI 8 HA antigens in all vaccine groups, but titers in the cHA groups exceeded the titers of the QIV group (FIG. 42A-42B). A difference in H9 titers, but not H2 or HI 8 titers was observed depending on the order of chimeric HA vaccination. Titers were higher in groups that received cH8/l followed by cH5/l, compared to groups that received cH5/l followed by cH8/l . [00560] cHA adjuvanted vaccine regimens induced higher anti-H2 IgG antibodies as compared to the non-adjuvanted vaccine regimen and the QIV control at both day 56 (28 days post-second immunization) and day 84 (28 days post-third immunization). The anti-H2 antibody responses detected are most likely due to an anti-Hl stalk domain cross-reactive antibody response rather than antibodies targeting conserved epitopes within the HA head domain of H2.

[00561] Similarly, cHA adjuvanted vaccine regimens induced higher anti-H9 and anti-Hl 8 IgG antibody responses as compared to the non-adjuvanted formulations and the QIV control. The anti-H9 or anti-Hl 8 antibody responses induced are expected to be predominantly HA stalk cross-reactive antibody responses. Of note, the order of the cHA (heterologous for the HA heads administration) tended to impact the magnitude of the induced anti-H9 antibody responses with CH8/1N1 followed by CH5/1N1 being slightly higher than the CH5/1N1 followed by CH8/1N1. Without being bound to any particular theory, this might be due to H8 and H9 both belonging to the H9-clade and being more antigenically related than H5, which could induce some level of HA head cross-reactive antibodies.

[00562] No induction of antibodies against H3 (group 2) was observed after vaccination with chimeric constructs that contain group 1 stalk domain (FIG. 44). This is consistent with the differences in stalk domains observed between influenza A group 1 and group 2 viruses and indicates that a multivalent chimeric vaccination approach will be necessary for a universal influenza virus vaccine.

(C) Anti-Neuraminidase antibody responses

[00563] Specific neuraminidase IgG antibody responses were also evaluated using an Nl specific ELISA assay in order to evaluate its potential implication in the protection on day 0, day 28 (28 days post- priming), day 54 (28 days post second immunization) and day 84 (28 days post third immunization (FIG. 11).

[00564] cHA adjuvanted vaccine regimens induced slightly higher anti-Nl IgG antibodies as compared to the non-adjuvanted vaccine regimen and the QIV control group.

(b) Fc-mediated functionality in vitro assay (ADCC reporter assay)

[00565] In order to evaluate the functionality of the anti-Hl stalk antibodies in vitro, Fc- mediated cytotoxicity was measured in an ADCC reporter assay with pooled sera from vaccinated mice at day 84 (FIG. 12 and FIG. 13). [00566] The two dose cHA immunization regimens elicited antibodies in QIV primed mice showing ADCC activity in vitro against wild-type A/HlNlpdm09 virus or stalk-conserved chimeric influenza virus cH6/lN5 (a reassortant influenza virus having an influenza A/Puerto Rico/8/1934 virus backbone, an A/mallard/Sweden/86/2003 (H12N5) NA, and a cH6/l chimeric HA, which has the HA globular head domain of A/mallard/Sweden/81/2002 (H6N1) HA and the HA stem domain of A/California/04/2009 (H1N1).

[00567] cHA adjuvanted vaccine regimens elicited higher ADCC reporter activity than any non-adjuvanted cHA vaccine candidates or the QIV control. No major differences in signal were measured between the sera from mice vaccinated with the non-adjuvanted cHA vaccine candidates and the QIV control. For all vaccine formulations, a higher ADCC-reported signal was detected against the HlNlpdm09 virus than cH6/lN5 virus. Of note, for cH6/lN5, the vaccine regimen CH8/1N1 - CH5/1N1 with ASOlB-derived formulation, induced higher ADCC activity compared to the CH8/1N1-CH5/1N1 regimen, suggesting a potential impact of the order of antigen immunization and adjuvant on the biological activity of the stalk-specific antibody responses.

(c) IgG2a IgGl ratios

[00568] To test if the differences observed in the ADCC reporter assay could be partially explained by a higher abundance of IgG2a (high Fc-receptor engagement) antibodies, compared to IgGl (low Fc-receptor engagement), HI stalk-specific antibody titers were measured with secondary antibodies specific to the IgG subtype. Serum samples from mice that received ASOl adjuvanted vaccines showed a higher ratio of IgG2a to IgGl antibodies by ELISA, which is indicative of a Thl-focused immune response that is often linked with induction of T cell responses. See FIG. 41 and FIG. 43.

(d) Serum transfer/viral challenge mouse studies

(A) Weight loss and survival after challenge with either HlNlpdm09 or CH6/1N5

[00569] The functional activity of the anti-Hl stalk domain antibodies induced by sequential immunization with cHA vaccine candidates was assessed by in vivo serum transfer into naive mice, followed by challenge with either wild-type A/Netherlands/602/2009 (A/HlNlpdm09) virus or stalk-conserved chimeric influenza virus cH6/lN5: pooled sera (Day 84) from mice vaccinated with the cHA-based regimen (non-adjuvanted or adjuvanted), or with the QIV control, or the PBS control were transferred into naive BALB/c mice. Two to five hours post- transfer, these mice were challenged with a cH6/lN5 virus or with a wild-type

A/Netherlands/602/2009 (A/HlNlpdm09) virus. Since sera from sequentially immunized mice are expected to react only to the HI stalk domain and not to the H6 head or the N5 NA, the

CH6/1N5 virus will allow the assessment of protection conferred by anti-Hl stalk domain antibodies only. Mouse body weights were monitored daily and survival following challenge was reported until day 14 or 16 (FIG. 14 and FIG. 15).

[00570] Sera from QlV-primed mice vaccinated with cHA adjuvanted vaccine regimens (either adjuvanted with AS03A or ASOlB-derived) when transferred into naive mice conferred protection following viral challenge. No weight loss and 100% survival was observed following challenge with a lethal dose of cH6/lN5 virus and 80 to 100 %> survival with moderate weight loss following challenge with a lethal dose of A/HlNlpdm09 virus. No differences between the adjuvants were observed. However, sera from QlV-primed mice vaccinated with cHA non- adjuvanted vaccine regimen induced only moderate (55 to 65% survival against cH6/lN5) to no protection (0% survival against A/HlNlpdm09) following lethal challenge. Sera from QIV- primed mice that received a second dose of QIV were protected up to 30% against cH6/lN5 and up to 70%) against HlNlpdm09. Following the cH6/lN5 challenge, weight loss was observed with sera from the QIV control regimen with moderate protection in contrast to the full protection observed with the sera from mice immunized with the adjuvanted cHA regimen, demonstrating the added value of the cHA sequential immunization approach and the need for an adjuvant system in the vaccine formulation to induce high levels of functional anti-Hl stalk antibodies able to confer protection against an heterologous viral strain (HI stalk conserved).

[00571] These results in the mouse passive transfer/viral challenge model demonstrate that sequential immunization with two cHA antigens (CH5/1N1 and CH8/1N1) adjuvanted with either AS03A or ASOlB-derived elicits potent functional anti-Hl stalk antibody responses that confer protection against HlNlpdm09 and cH6/lN5 influenza viruses.

6.2.4 DISCUSSION AND CONCLUSIONS

[00572] The cHA antigens (cH5/lNl and CH8/1N1) formulated with two Adjuvant Systems, either AS03A or ASOlB-derived, induce robust anti-Hl stalk domain IgG antibodies that bind antigenically distinct Group Al HA antigens in QlV-primed BALB/c mice and show in vitro ADCC activity. After serum transfer into naive mice, serum from mice vaccinated with adjuvanted vaccine regimen can prevent mortality and morbidity upon lethal challenge with two distinct influenza viruses, including a heterologous cH6/lN5 virus with an HA head domain and NA that the mice were not previously exposed to, but the conserved HI stalk domain. No major impact on the order of the antigen immunization (cH8/l or cH5/l) was observed in the different read-outs. However, greater ADCC activity was observed when CH8/1N1 was administered first in combination with ASOl . Also, the order of antigen administration seems to impact the levels of the anti-H9 antibody responses with CH8/1N1 followed by CH5/1N1 being slightly higher than the CH5/1N1 followed by CH8/1N1. Without being bound by any particular theory, this might be due to H8 and H9 both belonging to the H9-clade and being more antigenically related compared to H5. Serum samples from mice that received ASOl adjuvanted vaccines showed a higher ratio of IgG2a to IgGl antibodies by ELISA, which is indicative of a Thl-focused immune response that is often linked with induction of T cell responses. The induction of a higher T cell response indicates a more robust immune response to the influenza virus and potentially offers additional protection against a subsequent influenza virus infection.

[00573] Altogether these results support the proof of concept of the chimeric HA approach and demonstrate the need of an adjuvant system, either AS03A or ASOlB-derived, for optimal antibody induction.

6.2.5 REFERENCES

[00574] Nachbagauer, R., Kinzler, D., Choi, A., Hirsh, A., Beaulieu, E., Lecrenier, N., Innis, B. L., Palese, P., Mallett, C. P., & Krammer, F. A chimeric haemagglutinin-based influenza split virion vaccine adjuvanted with AS03 induces protective stalk-reactive antibodies in mice. Npj Vaccines 1, 16015 (2016).

[00575] Krammer, F., Pica, N., Hai, R., Margine, I. & Palese, P. Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies. J. Virol. 87, 6542-6550 (2013a).

[00576] Krammer, F. & Palese, P. Influenza virus hemagglutinin stalk-based antibodies and vaccines. Curr. Opin. Virol. 3, 521-530 (2013b).

[00577] Tong, S., Zhu, X., Li, Y., Shi, M., Zhang, J., Bourgeois, M, Yang, H., Chen, X., Recuenco, S., Gomez, J., Chen, L. M., Johnson, A., Tao, Y.,Dreyfus, C, Yu, W., McBride, R., Carney, P. J., Gilbert, A. T., Chang, J., Guo, Z., Davis, C. T., Paulson, J. C, Stevens, J., Rupprecht, C. E., Holmes,E. C, Wilson, I. A. and Donis, R. O. 2013. New world bats harbor diverse influenza A viruses. PLoS Pathog. 9: el003657.

6.3 EXAMPLE 3: IMMUNOGENICITY STUDY IN PRIMED AND UNPRIMED

RABBITS IMMUNIZED SUCCESSIVELY WITH MONOVALENT CHIMERIC HEMAGGLUTININ SUPRA-SEASONAL UNIVERSAL INFLUENZA VACCINE CANDIDATES: CH8/1N1 AND CH5/1N1 NON- ADJUVANTED, ADJUVANTED WITH AS03_A, OR ADJUVANTED WITH ASOIB-DERIVED

6.3.1 Objectives

[00578] The first objective of this Example was to determine if the monovalent influenza A virus Group 1 ("GrAl") cHA vaccine candidates (e.g., CH8/1N1 and CH5/1N1) when

administered as a two dose serial regimen with ASOlB-derived, AS03A, or non-adjuvanted induce anti-Hl stalk domain IgG antibodies in experimentally-primed NZW rabbits. The second objective of this Example was to determine if the same two dose regimen of the monovalent GrAl cHA vaccine candidates adjuvanted with ASOlB-derived, AS03A, or non-adjuvanted induce anti-stalk domain IgG antibodies in an unprimed NZW rabbit model. This second objective therefore helps to determine the added value of the priming to induce robust anti-Hl stalk domain IgG antibodies to guide future preclinical study designs.

6.3.2 Materials and Methods

(a) Overview of the study design

[00579] Groups of four rabbits each (two animals of each gender) received three

intramuscular ("IM") injections (500 μΕ/dose, equivalent to one human dose) on day ("D") 0, D 21, and D 35 with 15 μg HA/strain of QIV (non-adjuvanted), followed by 15 μg of CH8/1N1 and 15 μg of CH5/1N1, respectively, and formulated with either AS03A, ASOlB-derived, or without adjuvant (Groups 1-3) as described in Table 24. In addition, groups of four rabbits each (two animals of each gender) received two intramuscular injections (500 μΕ/dose, equivalent to one human dose) on D 21 and D 35 with 15 μg of CH8/1N1 and 15 μg of CH5/1N1, respectively, and formulated with either AS03A, ASOlB-derived or without adjuvant (Groups 4-6) as described in Table 24.

[00580] Table 24. Summary of the study design

^XQIV was produced in Dresden under the trade name of Fluarix Terta® (b) Investigational Products and Formulations Tested

[00581] See Section 6.2.1(c) above for investigation products and formulations tested in this Example.

(c) Ethical statement

[00582] The in vivo phase of the study was conducted in Marloie (CER group) Laboratory. The animal husbandry and study were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and the GSK Vaccines' policy on the care, welfare and treatment of animals. The ethical protocol CE/SANTE/G/001 was approved by the local Marloie CER ethical committee.

(d) Animal model

[00583] The study was conducted in NZW rabbits. At the beginning of the treatment period, the animals of both genders were approximately 10 weeks old and their body weight was between 2 kg and 2.5 kg. The animals were individually identified by an ear tattoo at the breeder's facility.

(e) Housing and Husbandry

[00584] The rabbits had free access to water and a maintenance diet. Consistent with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) global enrichment program, their environment included nesting material (Environment dry), and social housing was applied.

(f) Observations of clinical signs after vaccine administration

[00585] No abnormal observations were reported following administration of the QIV and cHA vaccine candidates.

(g) Immunological read-out: humoral responses - IgG binding antibodies by ELISA

[00586] The anti-Hl stalk domain ELISA using cH6/l recombinant antigen bound to the solid phase measured the IgG antibody in the rabbit sera directed against the conserved HA stalk domain.

[00587] Polystyrene 96-well ELISA plates (Nunc F96 Maxisorp cat 439454) were coated with 50 [iL of the recombinant cH6/l protein (recombinant HA with the head domain from H6 (A/mallard/Sweden/81/02) and an HI stalk domain (A/California/04/09 with the HA2 E42G stabilizing mutation) diluted to a concentration of 4 μg/mL in coating buffer (DPBS w/o Ca++ and Mg++ Bio Whittaker, cat BE17-512Q). The plates were incubated overnight at 4 °C. After incubation, the coating solution was removed and the plates were blocked with 100 μΕ of blocking solution (DPBS with 3% milk powder) per well for 1 hour at 37°C. The blocking solution was removed with four washes of PBS-Tween (PBS, 0.1% Tween-20, pH 7.4). The two-fold rabbit sera dilutions (in PBS + 0.1% Tween-20 + 1% BSA buffer) were added to the cH6/l-coated plates and incubated for 1 hour 30 min at 37°C. Horse radish peroxidase-labelled anti-rabbit IgG (whole molecule, Sigma, cat A8275) was used as the secondary antibody. Fifty microlitres of the secondary antibody diluted to a concentration of 1 :250 in PBS + 0.1% Tween- 20 + 1%) BSA buffer was added to each well and the plates were incubated for 1 hour at 37°C. The plates were washed four times with PBS-Tween and developed with 50 μΕ of o- Phenylenediamine (OPD, Sigma, cat P4664-50TAB) substrate per well for 20 minutes.

Enzymatic color development was stopped with 50 μΕ of 1M H2SO4 per well and the plates were read at an absorbance of 450/620 nm using the Versamax ELISA reader. Optical densities were captured and analysed using the SoftMaxvPro GxP v5.3 software. A standard curve was generated by applying a 4-parameter logistic regression fit to the reference standard results. Antibody concentration in the internal control serum or in samples was calculated by interpolation of the standard curve. The antibody titer of internal control serum or of the samples was obtained by averaging the values from dilutions that fell within the 20-80% dynamic range of the standard curve. Titers were expressed in EU (ELISA Units)/mL.

(h) Immunological read-out: Statistical Methods

[00588] The objective of this study was to generate a first set of information and no formal comparisons were performed. The sample size was selected based on a reasonable number of animals to confirm the ability of the vaccine candidates to induce a detectable anti-stalk domain IgG antibody response in NZW rabbits. The geometric mean titer (GMT) and 95% confidence interval (CI) for each group were determined with GraphPad Prism.

6.3.3 Results

(a) IgG Antibody Responses

[00589] To test the ability of cHA vaccine candidates to induce stalk domain-reactive antibodies, rabbits were either experimentally primed with QIV containing H1N1

A/Christchurch/16/10 to mimic pre-existing as in the human population and increase a baseline level of HA-stalk reactive antibodies that could then be boosted with the cHA vaccine candidates, or unprimed. Of note, some low background antibody responses (above the threshold of detection for some rabbits) were measured at D 0 before immunization which is infrequently detected in the sera of NZW rabbits. The anti-Hl stalk domain antibody responses, as determined by individual IgG ELISA titers measured for males and females, are provided in Table 25.

[00590] Table 25 : Anti-Hl stalk domain antibody responses: individual IgG ELISA titers measured for males and females

Anti-Hl stalk IgG (cH6/l) ELISA Titer (EU/ml)

Rabbit Animal

Formulations

Identification gender Day 0 Day 21 Day 38 Day 63

TBI Female 49 12920 23116

QIV(day 0) + TB2 Female 71 13771 14399

CH8/1N1 AS03A (day 21) + TB3 Male 69 6726 10722

CH5/1N1 AS03A (day 35) TB4 Male 4158 10462

GMT 18 46 8399 13901

TB5 Female 44 56 3838 3226

QIV (day 0) + TB6 Female 71 4430 3508

CH8/1N1 non-adjuvanted (day 21) + TB7 Male 80 3770 5247

CH5/1N1 non-adjuvanted (day 35) TB8 Male 62 2008 3893

GMT 23 67 3368 3899

TB9 Female 41 68 5474 7859

QIV (day 0)+ TB10 Female 46 4737 8604

CH8/1N1 AS01B -derived (day 21)+ TB11 Male 60 10515 15891

CH5/1N1 AS01B -derived (day 35) TBI 2 Male 18 2959 4146

GMT 22 43 5330 8170

TBI 3 Female N/A 7092 12357

TB14 Female N/A 5866 18280 TB15 Male 104 N/A 12515 20994

TB16 Male 47 N/A 4684 14233

GMT 35 N/A 7027 16118

TB17 Female N/A 187 1026

TB18 Female N/A 720 1910

CH8/1N1 non-adjuvanted (day 21)+

TB19 Male N/A 129 1336

CH5/1N1 non-adjuvanted (day 35)

TB20 Male 36 N/A 74 1217

GMT 21 N/A 189 1336

TB21 Female N/A 5232 8553

TB22 Female 150 N/A 3421 7173

CH8/1N1 AS01B -derived (day 21)+

TB23 Male 39 N/A 2958 11317

CH5/1N1 AS01B -derived (day 35)

TB24 Male 43 N/A 2065 15793

GMT 46 N/A 3234 10233

(A) QlV-primed rabbits (FIG. 16)

[00591] QIV priming elicited modest levels of anti-Hl stalk domain IgG antibody titers at 21 D post-immunization in most animals which is consistent with earlier preclinical

immunogenicity studies.

[00592] The two dose cHA regimen elicited robust anti-Hl stalk domain IgG antibody titers in QlV-primed animals when adjuvanted with either AS03A or ASOlB-derived at both D 38 (3 D post-third immunization) and D 63 (28 D post-third immunization).

[00593] Immunization with the non-adjuvanted cHA regimen elicited robust anti-Hl stalk domain IgG antibody titers on D 38, but those titers were 2.5 and 1.6-fold lower as compared to the titers induced by AS03A and ASOlB-derived adjuvanted formulations, respectively, whereas on D 63, those titers were 3.6 and 2.1-fold lower as compared to the titers induced by AS03A and ASOlB-derived adjuvanted formulations, respectively.

[00594] A trend for higher anti-Hl stalk domain IgG antibody titers was observed with the AS03A formulations compared to the ASOlB-derived formulations at both D 38 and D 63 (B) Unprimed rabbits (FIG. 17)

[00595] The two dose cHA regimen elicited robust anti-Hl stalk domain IgG antibody titers in unprimed animals when adjuvanted with either AS03A or ASOlB-derived at both D 38 (3 D post-second immunization) and D 63 (28 D post-second immunization) reaching comparable antibody titers as in QlV-primed animals.

[00596] Immunization with the non-adjuvanted cHA regimen elicited anti-Hl stalk domain IgG antibody titers on D 38, but those titers were 37.2 and 17.1-fold lower as compared to the titers induced by AS03A and ASOlB-derived adjuvanted formulations, respectively, whereas on D 63, those titers were 12.1 and 7.7-fold lower as compared to the titers induced by AS03A and ASOlB-derived adjuvanted formulations, respectively.

[00597] A trend for higher anti-Hl stalk domain IgG antibody titers was observed with the AS03A formulations compared to the ASOlB-derived formulations at both D 38 and D 63.

6.3.4 Discussion and Conclusion

[00598] The results show that specific anti-Hl stalk domain IgG responses were induced in all groups receiving either the full immunization regimen (QIV priming, CH8/1N1 and CH5/1N1) or only the cHA regimen (i.e., CH8/1N1 and CH5/1N1) without QIV priming. Higher anti-Hl stalk domain IgG antibody titers were measured in all groups immunized with the adjuvanted formulations than in the groups immunized with the non-adjuvanted formulations, with more pronounced differences in unprimed animals. A trend for higher anti-Hl stalk IgG antibody titers was observed in groups immunized with AS03A-adjuvanted formulations compared to ASOlB-derived adjuvanted formulations. The adjuvants had an important role in inducing high anti-stalk domain IgG antibody titers in the rabbit model confirming our previous observations in mice, and thus justifying the added value of our selected adjuvant systems. Of note, the QIV priming did not have an apparent impact on the anti-Hl stalk domain IgG antibody titers measured at the end of the immunization regimen at both D 38 and D 63 for the adjuvanted formulations whereas it increased the level of anti-stalk domain IgG antibodies elicited by the non-adjuvanted formulations.

[00599] In summary, this Example demonstrates that sequential immunization with cHA- based split virion vaccines can induce high titers of anti-stalk domain-specific IgG antibodies in experimentally-primed and unprimed NZW rabbits. 6.4 EXAMPLE 4: CLINICAL STUDY TO EVALUATE SUPRA-SEASONAL

UNIVERSAL INFLUENZA VACCINES (SUIVS) (UNADJUVANTED OR ADJUVANTED WITH AS03 OR AS01) IN HEALTHY ADULT SUBJECTS.

6.4.1 Introduction

[00600] This example relates to a Phase I/II, randomized, controlled, observer-blind, multi- center study to assess the reactogenicity, safety and immunogenicity of three supra-seasonal universal influenza vaccines (SUIVs) (unadjuvanted or adjuvanted with AS03 or AS01) administered as a 1 or 2-dose priming schedule followed by a booster dose 12 months postprimary vaccination in 18 to 39 year-old healthy subjects.

[00601] Current seasonal influenza vaccines show good efficacy when they are well-matched with the circulating virus strains. However, influenza viruses constantly change their surface glycoproteins that are the targets of most immune responses, allowing them to escape preexisting immunity, a process called antigenic drift. Therefore, seasonal influenza vaccines have to be reformulated and re-administered on an annual basis. In addition, novel viruses can appear at irregular intervals and cause influenza virus pandemics that can claim millions of lives.

[00602] Influenza A hemagglutinins (HAs) are phylogenetically divided into influenza A group 1 (HI, H2, H5, H6, H8, H9, HI 1, H12, H13, H16, H17 and H18) and influenza A group 2 (H3, H4, H7, H10, H14, and H15) based on sequence similarities. Protection from influenza viruses is usually correlated with antibodies that bind to the membrane distal head domain of the HA molecule, thereby blocking the virus from attaching to host cell receptors. However, the head domain has high plasticity and is the main site of antigenic drift.

[00603] The membrane proximal stalk domain of the HA is more conserved than the head domain and antibodies that target this domain have been shown to broadly neutralize influenza viruses across several subtypes. The stalk domain is immuno-subdominant compared to the head domain and is therefore usually not targeted by the immune system following exposure to influenza virus vaccines (Krammer F. Novel universal influenza virus vaccine approaches. Curr Opin Virol. 2016 Apr; 17: 95-103).

[00604] The proposed approach of the supra-seasonal universal influenza vaccine ("SUIV") described in this Example is to circumvent the immunodominance of the head domain by sequential exposure of the immune system to chimeric HAs (cHAs) that pair the globular head region of an exotic (for example, avian) HA with the HA stalk of a currently circulating seasonal influenza virus. The purpose of this approach is to boost pre-existing cross-reactive memory responses to the HA stalk without further boosting strain-specific responses against the head region of HA. This approach is combined with the use of a liposomal adjuvant (e.g., AS01).

6.4.1.1 Objectives

[00605] Primary objectives of the clinical study are: (1) to assess the reactogenicity and safety of each vaccine dose throughout the entire study period, in all study groups; and (2) to describe the anti-Hl stalk humoral immune response 28 days after each priming dose (1 or 2 dose(s)) in all study groups.

[00606] Secondary objectives of the clinical study are: (1) to evaluate the adjuvant effect of AS03 and AS01 on the humoral immune response after 1 and 2 priming dose(s) of

investigational SUIVs when compared to the non-adjuvanted formulations; (2) to describe the persistence of the anti-Hl stalk humoral immune response after each priming dose (1 or 2 dose(s)) in all study groups up to Month 14; (3) to describe the humoral immune response after a booster dose at Month 14; (4) to describe the breadth of the humoral immune response after each vaccination in all study groups; and (5) to describe the effect of the chimeric HA vaccination- sequence on the humoral immune response.

[00607] Tertiary objectives of the clinical study are: (1) to explore the cell-mediated immune responses (B-cells and T-cells) after each vaccination; (2) to explore the immune response against the IIV4 H1N1 and the HA head of CH5/1N1, CH8/1N1, cHl 1/lNl by hemagglutination inhibition (HI) assay; (3) to explore the anti-H3 stalk response (i.e. influenza A group 2); (4) to explore the immune response in terms of anti-NA antibodies after each vaccination; (5) to evaluate the occurrence of RT-PCR-confirmed influenza cases during the entire study period; (6) to explore the protective effect of the stalk-reactive antibodies induced by vaccination in a passive transfer challenge experiment in mice; (7) to develop and validate assays for

evaluation/characterization of the humoral and cellular immune responses to the investigational vaccines; (8) to explore the humoral immune response in term of anti-H9 full length HA serum antibodies; and (9) to explore anti-stalk antibody functionality (e.g. antibody-dependent cell- mediated cytotoxicity (ADCC), complement dependent lysis (CDL), antibody dependent cellular phagocytosis (ADCP) or glycoform analysis assays). 6.4.1.2 Rationale for the Study Design

[00608] Approximately 450 subjects 18-39 years of age are equally randomized in 10 different treatment groups and receive 2 or 3 doses of a monovalent influenza A group 1 SUIV or receive an annual quadrivalent inactivated seasonal influenza vaccine. Each SUIV contains a split inactivated influenza virus expressing a chimeric HA with the same stalk domain (HI stalk) at each dose, but a different exotic influenza A group 1 head. The antigen dose of 15 μg in each vaccine in this study is based on the standard antigen dose for inactivated seasonal influenza vaccines.

[00609] Without being bound by any particular theory, although it is assumed that a minimum of two priming doses followed by at least one booster dose may be required to induce long-term protection, the use of the adjuvant could significantly improve the anti-stalk immune response and induce adequate response with only one dose, especially in adults that have been previously exposed to the conserved HA stalk domain by natural exposure or vaccination.

[00610] Therefore, the vaccine regimen in the SUIV groups consists of sequential primary intramuscular immunization with one dose (Day 1) or 2 doses (Day 1 and Day 57) followed by a booster dose at Month 14 of a vaccine containing split inactivated influenza virus expressing a chimeric HA with at each dose the same stalk domain (HI stalk) but a different exotic head. The SUIV vaccine is adjuvanted with AS03A or ASO IE or non-adjuvanted. The interval of two months between the two priming doses was selected to ensure optimal priming in a setting where the vaccine can be administered the whole year round without time pressure for completion of vaccination in contrast with a seasonal or pandemic influenza vaccination setting. Without being bound by any particular theory, the booster dose at Month 14 may be necessary to obtain an adequate and persisting anti-stalk antibody response. In the control group, subjects are administered one dose of Fluarix Quadrivalent inactivated influenza vaccine (IIV4) on Day 1 and then re-vaccinated at Month 14 with the next year' s formulation.

[00611] The main purpose of this study is to assess the safety and the reactogenicity of each SUIV compared to IIV4. This study also evaluates the adjuvant effect of AS03A and ASO IE on the immune response when compared to the non-adjuvanted formulation. In addition, the immune response after 1 priming dose and after 2 priming doses is evaluated, as well as the immune response after a booster dose given 14 or 12 months after a one dose priming schedule or a 2-dose priming schedule, respectively. Since the vaccine sequence of the priming dose and the booster dose in the one dose-priming schedule groups varies, the effect of the chimeric HA vaccine sequence on the humoral immune response is assessed. Finally, the cell-mediated immune response after each vaccination and the protective effect in vivo of the anti-stalk antibodies is explored. Passive surveillance is put in place in order to capture the occurrence of RT-PCR-confirmed influenza cases during the entire study period.

6.4.2 Study Design

[00612] This clinical study is a Phase I/II, observer-blind, randomized, controlled, multicentric study with 10 parallel groups (a total of 450 subjects (45/group); see Table 26 for a description of the groups, see Table 27 for more details regarding the treatments {e.g., components, formulation, presentation, volume, and number of doses). Each group has a total of

45 subjects, 18 years to 39 years of age.

[00613] Table 26

*More detailed information regarding the treatments {e.g., components, formulation,

presentation, volume, and number of doses is provided in Table 27 below). [00614] Subjects in each group are treated as set forth in Figure 18. Briefly, subjects receive the vaccines indicated in Figure 18 on Day 1, Day 57, and Month 14. The vaccine is

administered to the subject intramuscularly ("FM") in the deltoid of the non-dominant arm. A description of the vaccines utilized is provided in Table 27. Blood is sampled on various days during the study as described in Section 6.4.2.1 below. During phase I, subjects are vaccinated one at a time until approximately 80 subjects are enrolled. If no safety issues are identified, then phase II is initiated. Interim analyses are performed after Day 85 and Month 14 + 28 days and the Final analysis is performed after Month 26. Passive surveillance for influenza-like illness ("ILI") is performed during the course of the study (i.e., between Day 1 and Month 26) as described in Section 6.4.2.1 below. Subjects are monitored for adverse events.

6.4.2.1 Sampling Schedule

[00615] Blood samples obtained \ on Days 1, 8, 29, 57, 64, and 85, Month 8, Month 14, Month 14 + 7 days, Month 14 +28 days, Month 20, and Month 26 are evaluated for safety assessment (see Section 6.4.3.4 below). In addition, blood samples are obtained during the screening period for subjects enrolled in the phase 1 portion of the clinical study.

[00616] Blood samples obtained on Days 1, 29, and 85, Month 8, Month 14, Month 14 + 7 days, Month 20, and Month 26 are evaluated for serology testing (see Section 6.4.3.2 below).

[00617] Blood samples obtained on Days 1 and 85, Month 14, and Month 26 are utilized in the passive transfer experiments described in Section 6.5 below.

[00618] Blood samples for cell-mediated immunity (CMI) assessment are drawn from a sub- cohort of approximately 50% of subjects at Days 1, 8, 29, 64, and 85, Month 14, Month 14 + 7 days, Month 14 + 28 days, and Month 26. The sub-cohort consists of the first Phase II subjects enrolled in pre-specified centers. See Section 6.4.3.3 below.

[00619] During the entire study period, nasal and throat swabs are collected as soon as possible (preferably within 24 hours, but not later than 7 days) after the onset of an ILI to test for influenza and/or other respiratory pathogens by RT-PCR (see Section 6.4.3.5 below). ILI is defined as at least one of these systemic symptoms: temperature (oral) greater than or equal to 37.8°C/98.6°F and/or, myalgia (widespread muscle ache); and at least one of these respiratory symptoms: cough and/or sore throat. Passive surveillance for ILI is carried out from Day 1 (after vaccine Dose 1) until the end of the study (Month 26). Subjects are instructed to contact the investigator/study staff as soon as they experience ILI symptoms. 6.4.2.2 Patient Population for Study

(a) Inclusion Criteria for Enrollment

[00620] All subjects satisfy all the following criteria at study entry: (1) a male or female between, and including, 18 and 39 years of age at the time of the first vaccination; (2) healthy subjects without acute or chronic, clinically significant pulmonary, cardiovascular, hepatic or renal functional abnormality, as established by medical history and clinical examination before first vaccination and laboratory screening tests (the latter being only applicable for subjects enrolled in Phase I); (3) subjects with no history of influenza vaccination within 6 months prior to first study vaccination and who are willing to forego any influenza vaccination during the entire study period; (4) female subjects of non-childbearing potential may be enrolled in the study; and (5) non-childbearing potential is defined as pre-menarche, current bilateral tubal ligation or occlusion, hysterectomy, bilateral ovariectomy or post-menopause. Female subjects of childbearing potential may be enrolled in the study, if the subject: has practiced adequate contraception for 30 days prior to first vaccination, and has a negative pregnancy test on the day of vaccination, and has agreed to continue adequate contraception during the entire treatment period and for 2 months after completion of the vaccination series (last vaccination at Month 14).

(b) Exclusion Criteria for Enrollment

[00621] The following criteria are checked at the time of study entry. If any exclusion criterion applies, the subject is not be included in the study: (1) use of any investigational or non- registered product (drug or vaccine) other than the study vaccines during the period starting 30 days before the first dose of study vaccines (Day -29 to Day 1), or planned use during the study period; (2) any medical condition that in the judgment of the investigator makes intramuscular injection unsafe; (3) chronic administration (defined as more than 14 days in total) of

immunosuppressants or other immune-modifying drugs during the period starting 6 months prior to the first vaccine dose; for corticosteroids, this means prednisone greater than or equal to 20 mg/day, or equivalent; inhaled and topical steroids are allowed; (4) administration of long-acting immune-modifying drugs (e.g. infliximab, rituximab) within 6 months before first vaccination (Visit 1), or planned administration any time during the study period; (5) planned

administration/administration of a vaccine not foreseen by the study protocol in the period starting 30 days before the first dose (Visit 1) up to the blood sampling at Day 85 (Visit 6) and in the period starting 30 days before booster vaccination at Month 14 (Visit 8) up to the blood sample at Month 14 + 28 days (Visit 10); (6) concurrently participating in another clinical study, at any time during the study period, in which the subject has been or will be exposed to an investigational or a non-investigational vaccine/product (pharmaceutical product or device); (7) previous vaccination against influenza within the 6 months preceding the first vaccination at Visit 1 or planned use of such vaccines during the study period; (8) history of vaccination with a (pre)pandemic influenza vaccine other than an HlNlpdm09 vaccine or history of laboratory- confirmed influenza infection other than seasonal or other than HlNlpdm09 influenza; (9) any confirmed or suspected immunosuppressive or immunodeficient condition, based on medical history and physical examination (no laboratory testing required); (10) history of or current autoimmune disease; (11) subjects diagnosed with excessive daytime sleepiness (unintended sleep episodes during the day present almost daily for at least one month) or narcolepsy; or history of narcolepsy in a subject's parent or sibling; (12) history of Guillain-Barre syndrome; (13) history of any reaction or hypersensitivity likely to be exacerbated by any component of the vaccines (including egg proteins); a history of anaphylactic-type reaction to consumption of eggs; or a history of severe adverse reaction to a previous influenza vaccine; (14)

hypersensitivity to latex; (15) acute disease and/or fever at the time of enrolment; fever is defined as temperature greater than or equal to 38.0°C / 100.4°F (preferably measured orally); subjects with a minor illness (such as mild diarrhea, mild upper respiratory infection) without fever may be enrolled at the discretion of the investigator; (16) administration of

immunoglobulins and/or any blood products during the period starting 3 months before the first dose of study vaccines or planned administration during the study period; (17) blood donation within 30 days before the first study blood sampling or planned blood donation within 30 days before and up to 30 days after any study blood sampling; (18) pregnant or lactating female; (19) history of chronic alcohol consumption and/or drug abuse as deemed by the investigator to render the potential subject unable/unlikely to provide accurate safety reports; and (20) female planning to become pregnant or planning to discontinue contraceptive precautions.

[00622] Additional exclusion criterion applicable for Phase I subjects includes: (1) hematological and/or biochemical parameters (complete blood cell count [red blood cells, white blood cells], white blood cells differential count [lymphocytes, neutrophils and eosinophils], platelets count or hemoglobin level, creatinine or blood urea nitrogen) outside the laboratory normal ranges, unless the laboratory abnormalities are considered not clinically significant by the investigator; and (2) liver enzymes (alanine aminotransferase [ALT] or aspartate

aminotransferase [AST]) outside of the normal laboratory ranges.

6.4.3 Materials and Methods

6.4.3.1 Vaccines, Products, and Treatment Names

[00623] Vaccines/products utilized in the study are provided in Table 27 below.

[00624] Table 27

MPL = Monophosphoryl Lipid A

aAS03A-like is obtained by dilution of the AS03 with PBS

AAS0 IE-like is obtained by dilution of the AS01_B with PBS

*QS-21 = Quillaja saponaria Molina, fraction 21 (Licensed by GSK from Antigenics Inc, a wholly owned subsidiary of Agenus Inc., a Delaware, USA corporation)

** After dilution and reconstitution

* * * After reconstitution

^#The strains depend on the World Health Organization recommendation for the Northern Hemisphere season (Dose 1 at Day 1) and the Northern Hemisphere season (booster at Month 14). 6.4.3.2 Humoral immune responses

[00625] Serological assays (quantification of antibodies by ELISA, microneutralization [MN] and hemagglutination inhibition [HI] assays) are performed using standardized procedures (see, e.g., Table 28). Further characterization of the vaccine-induced immune responses includes assessment of HLA-DRB 10401 and HLA-A201 alleles expression to evaluate the presence of HI stalk specific CD4+ and CD8+ T-cells using tetramer technology.

[00626] Table 28. Humoral immunity assays.

^$IIV4 HlNl strains are based on the World Health Organization recommendation for the 2017/2018 season (Dose 1 at Day 1) and the 2018/2019 season (booster dose at Month 14).

6.4.3.3 Cell-mediated immunity

[00627] T-cell, B-memory cell and plasmablast responses are evaluated using standardized procedures (see, e.g., Table 29).

[00628] Table 29

System Component Challenge Method Unit

PBMC = Peripheral blood mononuclear cells; JL-2 = interleukin-2; TNF-a = Tumor Necrosis Factor-alpha; IFN-γ = interferon-gamma; CD40-L = Cluster of Differentiation 40-Ligand; ICS = intracellular cytokine staining

6.4.3.4 Safety Assessment Assays: Hematology and Biochemistry

[00629] Hematology and biochemistry assays (see Table 30) are performed for safety assessment.

[00630] Table 30

*For white blood cell differential count.

¹ The Blood Urea Nitrogen (BUN)-to-creatinine ratio is to be calculated. 6.4.3.5 Molecular Biology (PCR tests)

[00631] At the onset of an ILI episode, nasal and throat swab specimens are taken. Each nasal and throat swab specimen are tested by RT-PCR for influenza and/or other respiratory virus infections as described in Table 31.

[00632] Table 31

6.4.3.6 Anti-stalk ELISA protocol

[00633] The prevalence of anti-stalk antibodies is measured by ELISA using reagents based on HA-group specific chimeric HAs. The chimeric HA consists of an exotic head domain on top of a vaccine-antigen stalk domain. For example, the cH6/l antigen has been constructed to measure anti-stalk antibodies against HI . Since humans are naive to the H6 head domain, reactivity measured with this substrate indicates reactivity to the HI stalk. Chimeric antigens are developed for the 3 HA groups (group Al, group A2 and group B) and are used in a classical ELISA. Briefly, the antigen is coated on 96 well plates. After blocking, the serum is added and sequentially diluted. After incubation and washing steps, a detection antibody is used to distinguish serum antibodies attached to the antigen and optical density measurement provides quantitative information about the amount of antibodies in the serum. Positive and negative controls are developed in addition of an antigen-specific standard.

[00634] The breadth of the immune response within the group 1 HA subtypes is measured using a full length HA ELISA.

6.4.3.7 Microneutralization (MN) assay protocol:

[00635] The functionality of the stalk-reactive antibodies is evaluated by MN assays developed using chimeric viruses. As done for the ELISA, the objective is to avoid interference caused by antibodies directed against HA head but also caused by antibodies directed against NA; for that purpose viruses expressing an HA with an HA group-specific stalk and an HA head domain and NA to which humans are naive are used. Briefly, measurements are conducted on thawed frozen serum samples. Samples are heat inactivated for 30 min at 56°C. A standardized amount of virus is mixed with serial dilutions of serum and incubated to allow binding of the antibodies to the virus. A cell suspension, containing a defined amount of Madin-Darby Canine Kidney (MDCK) cells is then added to the mixture of virus and antiserum and incubated at 35°C (plus or minus 2°C). After the incubation period, virus replication is visualized (by

hemagglutination of red blood cells or OD reading) and a neutralization titer is calculated between the highest serum dilution able to totally neutralize the virus and one of the next serum dilution where viruses remain detectable. Each serum sample is tested once.

6.4.3.8 Hemagglutination inhibition (HI) assay protocol:

[00636] HI antibody titers are determined using the method derived from the WHO Manual on Animal Influenza Diagnosis and Surveillance, WHO/CD S/CSR/NCS/2002.5, which is incorporated by reference herein in its entirety.

[00637] Measurements are conducted on thawed frozen serum samples with a standardized and comprehensively validated micro-method using 2 hemagglutinating units (2 HAU) of the appropriate antigens and a 0.45% fowl erythrocyte suspension. Non-specific serum inhibitors are removed by heat treatment and receptor-destroying enzymes.

[00638] Starting with an initial dilution of 1 : 10, a dilution series (by a factor of 2) is prepared up to an end dilution of 1 : 10240. The titration end-point is taken as the highest dilution step that shows complete inhibition of hemagglutination. All assays are performed in duplicate. The usual cut-off value is 10 1/DIL.

6.4.3.9 ELISPOT (memory B cell detection assay):

[00639] The B-cell ELISPOT allows the quantification of antigen-specific memory B-cells. These cells are responsible of long term (humoral) memory and will be the cells recalled during an infection subsequent to vaccination. This assay is designed to evaluate the frequency (per million memory B-cells) of HA stalk specific memory B-cells from peripheral blood samples.

[00640] The protocol is adapted from the assay developed by Crotty et al. (Crotty et al., Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J Immunol Methods. 2004 Mar; 286 (1-2): 111-22), and involves the incubation of PBMC that have been differentiated into antibody secreting cells in nitro-plates coated with either the antigen of interest (for the detection of antigen-specific memory B-cells) or anti-human Ig (for the detection of total memory B-cells). A conventional immuno-enzymatic procedure (Crotty et al. Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J Immunol Methods. 2004 Mar; 286 (1-2): 111-22. ) is applied to detect antibody/antigen spots enumerating memory B-cells and the results are expressed as the frequencies of antigen- specific memory B-cells within the total memory B-cell population.

6.4.3.10 Flow Cytometry B-cells (FCB) for plasmablasts detection

[00641] The FCB assay has been developed to allow detection of HA stalk specific plasmablasts. Upon vaccination or natural challenge, these differentiated B-cells are transiently present in the periphery (peak at Day 7 post-antigen encounter) and are the cells producing the antibodies. The FCB assay has been set up to detect the cells producing the anti-HA stalk antibodies that the SUIV is designed to induce.

[00642] The PBMC are thawed and immuno-stained for surface markers allowing phenotypic identification of the plasmablasts (CD14- / IgD- / CD20- / CD19+ / CD38+). Cells are then permeabilized and immune-stained with a specific biotinylated probe (here the chimeric HA protein cH6/l) which will bind to the cognate antibodies (here the anti-HA stalk antibodies) within the plasmablasts. The probe is then detected with a fluorescently labelled streptavidin, and analysis performed by multi-parametric flow cytometry. Results are expressed as frequencies of antigen specific plasmablasts per million PBMC. 6.4.3.11 T-cell detection by Intracellular Cytokine Staining (ICS) assay:

[00643] This assay is applied to measure the frequency of antigen-specific T-lymphocytes in peripheral blood. CD4+ T-lymphocytes are critical helper cells supporting the differentiation of antibody secreting cells (B-cells). CD8+ T-lymphocytes are direct effector cells able to kill virus-infected cells.

[00644] Blood samples are collected by venipuncture and PBMCs are prepared by

centrifugation onto a LymphoprepTM cushion within 24 hours following collection. PBMC suspensions are stored in liquid nitrogen until analysis. To measure T-cell responses elicited by the vaccine candidate, samples are thawed and stimulated with relevant antigen (synthetic peptides covering the HA stalk domain). Re-stimulated cells are then immunostained for surface markers such as CD3, CD4 and CD8 followed by cell permeabilization and immunostaining for effector molecules (cytokines/activation marker) such as IL-2, IFN-γ, T F-α and CD40L (additional effector molecules could also be explored). Analysis is performed by

multiparametric flow cytometry, and the results are expressed as frequencies of CD4+ (or CD8+) T-cells producing various combinations of the cytokine/activation markers assessed per million CD4+ (or CD8+) T-cells.

6.4.3.12 RT-qPCR assay for influenza detection:

[00645] Viral RNAs extracted from the clinical sample are amplified and detected using Flu- A and Flu-B specific primers and probes (designed in the Matrix gene). Viral Load values are quantified and the sample is considered positive when the measured Viral Load is equal to or above the assay cut-off. Several controls are included throughout the process, both at the extraction and RT-PCR steps to monitor the extraction and RT-PCR efficiencies and any potential contamination that may occur during each run.

6.4.3.13 RT-PCR assay for influenza A H1N1 and A H3N2 typing:

[00646] A RT-PCR allowing the identification of Flu-A/H1N1 and Flu-A/H3N2 is performed on the nucleic acids generated for Flu-A and Flu-B detection. Specific primers designed in the HA gene of Flu-A/H1N1 and Flu-A/H3N2 are used to perform the discrimination. In addition, Flu-A/H1N1 and Flu-A/H3N2 RT-PCR positive controls corresponding to the RNA from reference strains are evaluated in parallel with the samples. No template controls are also used to monitor any potential contamination during each RT-PCR run. 6.4.3.14 RT-qPCR assay for RSV detection:

[00647] Viral RNAs extracted from the clinical sample are amplified and detected using RSV- A and RSV-B specific primers and probes designed in the N gene encoding the nucleocapsid protein. Viral load values are quantified and the sample is considered positive when the measured viral load is equal to or above the assay cut-off. Several controls are included throughout the process, both at the extraction and RT-PCR steps to monitor the extraction and RT-PCR efficiencies and any potential contamination that may occur during each run.

6.4.3.15 Multiplex RT-PCR assay for viral pathogen detection:

[00648] A qualitative PCR multiplex assay is used for the detection and identification of multiple respiratory virus nucleic acids in nasal and throat swabs from individuals suspected of respiratory tract infections. The following virus types and subtypes are identified in the assay: Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Parainfluenza 4 virus, Human Metapneumovirus, Rhinovirus, Enterovirus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Coronavirus L63, and Human Bocavirus.

[00649] Following total nucleic acids extraction, viruses are detected by multiplex real-time RT-PCR assays targeting the above mentioned viruses. A comparative analysis of the

fluorescence intensities of each target is performed to detect the viruses present in the sample. Several controls are included throughout the process, both at the extraction and RT-PCR steps to monitor the extraction and RT-PCR efficiencies and any potential contamination that may occur during each run.

6.5 EXAMPLE 5: SERUM PASSIVE TRANSFER/VIRUS CHALLENGE

EXPERIMENT IN BALB/C MICE FROM ADULT SUBJECTS INVOLVED IN THE FLU D-SUIV-ADJ-001 STUDY COHORT

[00650] The in vivo protective effect of transferring pooled adult human serum from subjects in the clinical study described in Section 6.4 above to mice and subsequently challenging the mice with chimeric head-stalk HA virus, for example cHx/lNy virus (Hx = possibly H6/1, Ny = possibly N5) or circulating H1N1 (possibly H1N1 strain contained in the IIV4 control annual vaccines in accordance with WHO recommendation) is assessed in terms of the following endpoints: (1 A) Survival over 14 days post-challenge (day of death or euthanasia for weight loss > 25% baseline body weight) in groups of 10 to 25 mice/human-group/timepoint (number of mice is decided upon the pathogenicity of the challenge virus in mice); (IB) Mean weight loss (change from baseline over 14 days post-challenge) in groups of 10 to 25 mice/human- group/timepoint; and (1C) Lung virus titer in pfu^g [loglO fold-change (Day 1 - Day 85), (Day 1 - Month 14), (Day 1 - Month 26)] on Day 3 and Day 6 post-challenge in subset of 5 mice for each timepoint.

[00651] The association between post-transfer ELIS A titer of human IgG to the challenge viruses (anti-stalk IgG) at Day 3 and Day 6 and the post-challenge outcome is explored with the object to measure of the post-transfer geometric mean ELIS A titer of human IgG to cH6/lNx virus and human IgG to A/HlNlpdm09 virus in blood collected from mice receiving either one of the serum pools (Day 1, Day 85, Month 14 and Month 26) upon challenge and at Day 3 and Day 6 post-transfer. The correlation between post-challenge endpoints 1 A, IB, and 1C and (a) proportion of survival over 14 days post-challenge, (b) mean weight loss, and (c) geometric mean lung virus titer is evaluated.

6.5.1 Methods

6.5.1.1 Viruses

[00652] Table 32

[00653] Both viruses are pre-assessed for mouse lethality in LD50 experiments (with and without presence of adult human serum pools). The volume and route of inoculation is 0.05 mL intra-nasal/intra-tracheal administered to anesthetized mice. 6.5.1.2 Human serum pools

[00654] Serum pools are created using all residual serum samples with sufficient volume to furnish an aliquot of an equal volume across the groups and the time points Day 1, Day 85, Month 14 and Month 26 from the subjects in the clinical study described in Section 6.4 above.

6.5.1.3 Passive transfer experimental method

[00655] Twenty to 35 mice per timepoint per virus challenge are transferred with a standard amount of undiluted human pooled serum (volume within the range 150-250 μΐ.): (35 mice x 2 virus challenges x 4 timepoints = 280 mice in total per human group). However, depending on the lethality of the virus or if sufficient serum volumes are not available, the number of mice is reduced to as low as 10 mice per timepoint and virus for the survival monitoring and 10 for the lung virus titer assays. Also, most likely all the human group serums are tested but some groups are excluded depending on the results obtained, as the clinical trial described in Section 6.4 above progresses and in accordance with the animal ethical 3R's principles.

[00656] Two hours post serum transfer, the mice are sedated and: (1) Blood is collected for determination of post-transfer human IgG titers by ELISA; and (2) challenged with 5 x LD50 delivered by the IN/IT route (or a lower dose selected to provide a level of weight-loss and lethality using Day 1 serum that could be reduced by a 3-10 fold increased level of anti-HA stalk antibodies present in post-vaccination serum).

[00657] Mice are monitored daily for 14 days for weight-loss and are euthanized if they lose > 25% of their initial body weight or if any human endpoints defined in the ethical protocol described in Section 6.4 above are met.

[00658] On Day 3 and Day 6 post-infection, 5 mice per timepoint/virus are euthanized to assess viral lung titers (5 mice per timepoint/virus x 2 harvesting days x 5 timepoints x 2 virus; 100 mice in total).

[00659] Weighing of the mice and lungs processing is performed in an open fashion. Viral plaques are counted by a blinded technician.

[00660] Lung suspensions are made by homogenization in PBS and frozen at -80°C for later plaque assay using a standard method. 6.5.1.4 Statistical analytical plan

[00661] Primary objective is defined as the improved survival after challenge of any vaccine groups (mice receiving human pooled serum from chimeric HA- approach groups) compared to control group (mice receiving human pooled serum from IIV4 group), measured at each time point. Additional exploratory testing might be added to the study, including potential correlation between post-transfer titers and survival or correlation between lung-titers at Day 3 or Day 6 and survival.

6.6 EXAMPLE 6: DESIGN AND CHARACTERIZATION OF CHIMERIC HA

[00662] Through replacement of dominant antigenic sites on the head of the influenza B virus hemagglutinin (HA) with sequences from exotic influenza A virus strains (FIGS. 26 and 27), chimeric HAs have been generated that are easily incorporated into functional viruses through reverse genetics. Initial in vitro characterization of the chimeric HA and viruses expressing these HA, including growth analysis (FIG. 36) and surface expression (FIG. 37) were performed. Additionally, through replacement of antigenic sites, a decrease in HI endpoint titer in viruses with chimeric HA compared to wild type HA was apparent (FIG. 38), emphasizing that antigenic sites have functionally been changed.

6.6.1 Materials and Methods

[00663] Structural Overlays: Structural overlays were performed using Pymol software.

[00664] Sequence Alignments: Sequence alignments of HAs to determine corresponding loop/helix sequences were performed using BioEdit software.

[00665] Plaque Assay: Step 1 : 2x media was prepared for agarose overlay (50 mL total volume: 25 mL 2x MEM, 9ML WFI water, 660 μL· 7.5% sodium bicarbonate, 500 μL· 1% dextran) and stored in water bath at 37°C. Step 2: Viral dilutions (dilutions used depend on sample and expected titer) were performed as follows: (a) PBS/0.3% BSA + 1%

penicillin/streptomycin was combined with Ca²⁺ /Mg²⁺, and 450 μΕ was added to each tube for dilutions; (b) 50 μΕ of virus was added to the first tube, the tube was mixed, and then 50 μΕ from the tube was added to the next tube, etc. Step 3 : Pre-plated cells were washed in 6-well plates with 1 mL PBS. Step 4: 300 μΕ virus dilution (from step 2) was added to each well; most dilute to least dilute to avoid concentration variance. Step 5: Adsorption: cells were incubated at 33°C or 37°C (depending on the virus) for 1 hour; and were rocked every 10-15 minutes. Step 6: When adsorption was almost finished, 2% agarose was heated in microwave and 15 mL was added to media from step 1. Step 7: TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) treated trypsin (lmg/mL) at 1/1000 dilution was added to media from step 1 (resulting concentrating is 1 ug/mL). Step 8: agarose/media/trypsin mixture was poured evenly on 6 well plates (2mL/well) and allowed to harden without disturbing overlay. Plates were stored at 33°C for 72 hours or 37°C for 48 hours depending on the virus.

[00666] Surface staining: MDCK cells were plated the day prior to infection. Cells were infected with indicated viruses at a multiplicity of infection (MO I) of 5 without TPCK-trypsin and incubated at 33 degrees Celsius. 17 hours post-infection, cells were fixed with methanol- free 4% PFA for immunofluorescence surface staining using the indicated anti-influenza B HA cross-protective human/mouse monoclonal antibodies and anti-influenza B virus HA polyclonal mouse serum. Secondary Alexa-Fluor 488 anti-human or anti-mouse antibody was used.

Images were taken using Zeiss LSM 880 confocal microscope.

[00667] Growth curves in eggs: 10-day old embryonated chicken eggs were infected with 500 plaque forming units/egg of wild type influenza B/Malaysia/2506/04 MA virus or influenza B/Malaysia/2506/04 MA virus expressing chimeric HA. Growth curves were performed in triplicate. Allantoic fluids were harvested at 8 hours, 24 hours, 48 hours, and 72 hours post infection. Plaque assays were performed on MDCK cells as described above to determine virus titers.

[00668] HI assay: Mouse and ferret sera were raised against wild type influenza B virus strain B/Yamagata/16/88 to acquire hemagglutination inhibition (HI) reactivity. Prior to performing HI assay, any serum that was to be used as antibody for the HI assay was pretreated with Receptor-Destroying Enzyme (BioWhittaker, Walkersville, MD), 100 units/mL, prepared according to manufacturer's instructions. This resulted in serum being at a 1 : 10 dilution starting concentration (at the highest) for the HI assay. The HA assay was performed on test virus and positive control virus and the dilution that yielded 8 HAU (4 positive wells) was determined. This standardized virus was used for the assay. To find the dilution that would yield 4 wells, the last positive well was divided by 8. This provided the dilution factor, i.e., if the last dilution to have positive agglutination was 128 HAU, then to obtain 8 HAU the following calculation was performed: 128/8=16. To make 8 HAU, a 1 : 16 dilution was performed. In order to make sure the titer of the new dilution was 8 HAU, back titration was performed.

[00669] The following lanes of controls were included on the plate: (A) lane with PBS + virus (no antibody); (B) lane with 50 μΐ_^ PBS (no antibody) and no virus. To plate, 50 μΐ_^ antibody was added to column 1 and 25 μΐ_^ PBS was added to other wells. 25 μΐ_^ from the first column to the second column (2 fold dilutions of antibody) was transferred until the last columns with PBS alone and no antibody. Each well had 25 μΐ_^ of the dilution. 25 μΐ_^ of the appropriate virus was added to all other wells (either test virus, or positive control virus). PBS was added for "no virus" control. Virus and antibody were pre-incubated at room temperature for 30 minutes. 50 uL of 0.5% RBCs (turkey) in PBS was added as usual for hemagglutination assay. The plates were kept at 4°C for 30 minutes. Pictures of the results were captured with a scanner.

[00670] Generation of chimeric HAs: Design of the HA constructs was based on determining corresponding residues for each loop or helix that was described by Wang et al , 2008, Journal of Virology, 82(6):301 1-3020. Corresponding residues were determined by protein structural alignments as well as sequence alignments.

[00671] For generation of chimeric HA constructs, HAs were synthesized. Alternatively, the HA segment was generated by performing PCR on previously constructed plasmids and by using these PCR products (with at least 15 nucleotides overlap between each product) during the Infusion® (Clontech) cloning reaction. To insert the HA segment into its vector, plasmid pDZ was linearized with SAPI restriction enzyme. Each HA fragment contained 15 nucleotide overlap with the pDZ linearized vector. This overlap region was included during design for synthesis of the HA, or was artificially added on by adding this sequence to the primers amplifying from the non-coding regions of the HA during PCR.

[00672] In-fusion® cloning reactions were performed with the generated HA products and linearized pDZ by using the reaction buffer as described by the manufacturer Clontech.

Amounts of PCR fragments of HA, synthesized HA, or linearized pDZ vector were adjusted to have equal molar ratios (calculated based on fragment length). The In-fusion® reaction incubated in a PCR machine at 50 degrees Celsius for 15 minutes then was removed. The resulting cloning product was transformed into Stellar cells and was plated in LB plates containing ampicillin. Resulting colonies were screened for presence of the correct HA insertion sequence and could be grown up and purified as mini-, midi-, or maxi- preps of the DNA. This resulted in the final plasmid product. The concentration of the final product was determined by methods such as using a Nanodrop.

6.6.2 Results

[00673] To generate influenza B virus chimeric HAs that could serve as universal vaccine candidates, key antigenic sites (the 120 loop, the 150 loop, the 160 loop, and/or the 190 helix (see Wang et al, 2008, Journal of Virology, 82(6):3011-3020 for a description of the 120 loop, 150 loop, 160 loop, and 190 helix)) in the influenza B virus globular head domain of influenza B/Yamagata/16/88 virus HA were modified based on antigenic sites from the globular head domain of influenza A viruses of the H5, H8, HI 1, or HI 3 subtypes (influenza

A/Vietnam/1203/04 virus (HALo) (H5), influenza A/Mallard/Sweden/24/2002 virus (H8), influenza A/northern shovel er/Netherlands/18/99 virus (HI 1)), or A/black headed

gull/Sweden/ 1/99 (H13), respectively) (referred to as mH5/B, mH8/B, mHl 1/B, and mH13/B, respectively, chimeric HAs) (FIG. 26 and FIG. 27).

[00674] To generate the mH5/B chimeric HA for rescue in an influenza B/Malaysia/2506/04 MA virus backbone, amino acid residues relating to the 120 loop, 150 loop, 160 loop, and 190 helix of the influenza B/Yamagata/16/88 virus HA globular head were modified based on amino acids from the influenza A/Vietnam/1203/04 (HALo) (H5) virus globular head domain (FIGS. 28 and 29). In particular, the amino acid sequences from of the influenza B/Yamagata/16/88 virus HA 120 loop (TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108)), 150 loop (PNVTSRNG (SEQ ID NO: 123)), 160 loop (RDNKTA (SEQ ID NO: 110)), and 190 helix (NKNQMKN (SEQ ID NO: 112)) were replaced with amino acid sequences FIP,

KIQL S TKN VIN AEH APGGP YRL (SEQ ID NO: 109), PYQGKSS (SEQ ID NO: 124),

KKNSTY (SEQ ID NO: 111), and NDAAMQT (SEQ ID NO: 113), respectively, based on amino acid sequences of the influenza A/Vietnam/1203/04(HALo) (H5) virus globular head domain. Influenza B/Malaysia/2506/04 MA virus was used as the backbone (i.e., the PB2, PB1, PA, NP, NA, M, and NS are from influenza B/Malaysia/2506/04 MA virus) to rescue virus expressing this chimeric HA construct. Additionally, the glutamic acid (E) amino acid at position 156 of the immature influenza B/Yamagata/16/88 virus HA was substituted with a lysine (K) (i.e., an E156K mutation) in the chimeric HA. This virus was successfully rescued, having a stock titer of 6.95 x 10⁸ PFU/mL. This virus was slightly attenuated when grown in 10- day old embryonated eggs (500 PFU/egg inoculant) at 33 degrees Celsius, as compared to a control influenza B/Malaysia/2506/04 MA virus (FIG. 36A). The peak titer p value was 0.0005.

[00675] To generate the mH8/B chimeric HA for rescue in an influenza B/Malaysia/2506/04 MA virus backbone, amino acid residues relating to the 120 loop, 150 loop, 160 loop, and 190 helix of the influenza B/Yamagata/16/88 virus HA globular head were modified based on amino acids from the influenza A/Mallard/Sweden/24/2002 virus (H8) globular head domain (FIGS. 30 and 31). In particular, the amino acid sequences of the influenza B/Yamagata/16/88 virus HA 120 loop (TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108)), 150 loop

(PNVTSRNG (SEQ ID NO: 123)), 160 loop (RDNKTA (SEQ ID NO: 110)), and 190 helix (NKNQMKN (SEQ ID NO: 112)) were replaced with amino acid sequences HIP,

RIRLSTYNVINAETAPGGPYRL (SEQ ID NO: 125), NASTGGQS (SEQ ID NO: 126), KKKADTY (SEQ ID NO: 127), and ADAKMQT (SEQ ID NO: 128), respectively, based on amino acid sequences of the influenza A/Mallard/Sweden/24/2002 virus (H8) globular head domain. Influenza B/Malaysia/2506/04 MA virus was used as the backbone (i.e., the PB2, PB1, PA, NP, NA, M, and NS are from influenza B/Malaysia/2506/04 MA virus) to rescue virus expressing this chimeric HA construct. Additionally, the glutamic acid (E) amino acid at position 156 of the immature influenza B/Yamagata/16/88 virus HA was substituted with a lysine (K) (i.e., an E156K mutation) in the chimeric HA. This virus was successfully rescued, having a stock titer of 1.16 x 10⁹ PFU/mL. This virus was slightly attenuated when grown in 10- day old embryonated eggs (500 PFU/egg inoculant) at 33 degrees Celsius, as compared to a control influenza B/Malaysia/2506/04 MA virus expressing an HA comprising the influenza B/Malaysia/2506/04 MA virus (FIG. 36B). The peak titer p value was 0.0004.

[00676] To generate the mHl 1/B chimeric HA for rescue in an influenza B/Malaysia/2506/04 MA virus backbone, amino acid residues relating to the 120 loop, 150 loop, 160 loop, and 190 helix of the influenza B/Yamagata/16/88 virus HA globular head were modified based on amino acids from the influenza A/northern shovel er/Netherlands/18/99 virus (HI 1) globular head domain (FIGS. 32 and 33). In particular, the amino acid sequences of the influenza

B/Yamagata/16/88 virus HA 120 loop (TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108)), 150 loop (PNVTSRNG (SEQ ID NO: 123)), 160 loop (RDNKTA (SEQ ID NO: 110)), and 190 helix (NKNQMKN (SEQ ID NO: 112)) were replaced with amino acid sequences LIP, KIEL S T SNVTN AE V AP GGP YRL (SEQ ID NO: 129), PFGSSNS (SEQ ID NO: 130), HQSGTY (SEQ ID NO: 131), and TTLKMHQ (SEQ ID NO: 132), respectively, based on amino acid sequences of the influenza A/northern shoveler/Netherlands/18/99 virus (HI 1) globular head domain. Influenza B/Malaysia/2506/04 MA virus was used as the backbone (i.e., the PB2, PB1, PA, NP, NA, M, and NS are from influenza B/Malaysia/2506/04 MA virus; see SEQ ID NOs: 161, 162, 163, 164, 165, 166, and 167 for the nucleotide sequences) to rescue virus expressing this chimeric HA construct. Additionally, the glycine (G) amino acid at position 250 of the immature influenza B/Yamagata/16/88 virus HA was substituted with a glutamic acid (E) (i.e., a G250E mutation) in the chimeric HA. This virus was successfully rescued, having a stock titer of 1.42 x 10⁸ PFU/mL. This virus was slightly attenuated when grown in 10-day old embryonated eggs (500 PFU/egg inoculant) at 33 degrees Celsius, as compared to a control influenza B/Malaysia/2506/04 MA virus (FIG. 36C). The peak titer p value was 0.2310.

[00677] Table 33 Nucleic acid sequences encoding Influenza B/Malaysia/2506/04 MA virus PB1, PA, NP, NA, M, and NS.

Sequence Sequence (SEQ ID NO:)

Descriptio

n

tcaaagaggcagatataaccccagcacatggtccagtaaagaaaatggactacgatgcggtgtctggaactcatagtt ggagaaccaaaagaaacagatctatactaaacactgatcagaggaacatgattcttgaggaacaatgctacgctaagt gttgcaacctatttgaggcctgttttaacagtgcatcatacaggaagccagtgggtcaacatagcatgcttgaggctatg gcccacagattaagaatggatgcacgattagattatgaatcaggaagaatgtcaaaggatgattttgagaaagcaatgg ctcaccttggtgagattgggtacatataagcttcgaagatgtctatggggttattggtcatcattgaatacatgcgatacac aaa (SEQ ID NO: 161)

Nucleotide ttcaagatgacattggccaaaattgaattgttaaaacaactgctaagggacaatgaagccaaaacagttttgaagcaaa sequence of caacggtagaccaatataacataataagaaaattcaatacatcaaggattgaaaagaatccttcactaaggatgaaatg mouse ggccatgtgttctaattttcccttggctctaaccaagggcgacatggcaaatagaatccccttggaatacaaaggaatac adapted aacttaaaacaaatgctgaagacataggaaccaaaggccaaatgtgctcaatagcagcagttacttggtggaatacat Influenza atggaccaataggagatactgaaggtttcgaaagggtctacgaaagcttttttctcagaaaaatgagacttgacaatgcc B/Malaysia acttggggccgaataacttttggcccagttgaaagagtgagaaaaagggtactgctaaaccctctcaccaaggaaatg /2506/2004 cctccagatgaggcgagcaatgtgataatggaaatattgttccctaaagaagcaggaataccaagagaatccacttgg vims PB2 atacatagrgaactgataaaagaaaaaagagaaaaattgaaaggaacaatgataactccaatcgtactggcatacatg cttgaaagagaactggttgctcgaagaagattcttgccagtggcaggagcaacatcagctgagttcatagaaatgctac actgcttacaaggtgaaaattggagacaaatatatcacccaggagggaataaattaactgagtctaggtctcaatcaat gatagtagcttgtagaaaaataatcagaagatcaatagtcgcttcaaacccactggagctagctgtagaaattgcaaac aagactgtgatagatactgaacctttaaagtcatgtctggcagccatagacggaggtgatgtagcttgtgacataataag agctgcattaggactaaagatcagacaaagacaaagatttggacggcttgagttaaaaagaatatcaggaagaggatt caaaaatgatgaagaaatattaatagggaacggaacaatacagaagattggaatatgggacggggaagaggagttc catgtaagatgtggtgaatgcaggggaatattaaaaaagagtaaaatgaaactggaaaaactactaataaattcagcca aaaaggaggacatgagagatttaataatcttatgcatggtattttctcaagacactaggatgttccaaggggtgagagga gaaataaattttcttaatcgagcaggccaacttttatctccaatgtaccaactccaacgatattttttgaatagaagcaacg acctttttgatcaatgggggtatgaggaatcacccaaagcaagtgaactatatgggataaatgaatcaatgaatgcatct gactatacattgaaaggggttgtagtgacaagaaatgtaattgacgactttagctctactgaaacagaaaaagtatccat aacaaaaaatcttagtttaataaaaaggactggggaagtcataatgggagctaatgacgtgagtgaattagaatcacaa gcacagctgatgataacatatgatacacctaagatgtgggaaatgggaacaaccaaagaactggtgcaaaacacttat caatgggtgctaaaaaacttggtaacactgaaggctcagtttcttctaggaaaagaggacatgttccaatgggatgcatt tgaagcatttgagagcataattcctcagaagatggctggtcagtacagtggatttgcaagagcagtgctcaaacaaatg agagaccaggaggttatgaaaactgaccagttcataaagttgttgcctttttgtttctcaccaccaaaattaaggagcaat ggggagccttatcaattcttaaaacttgtattgaaaggaggaggggaaaatttcatcgaagtaaggaaagggtcccctc tattttcctataatccacaaacagaagtcctaaccatatgcggcagaatgatgtcattaaaagggaaaattgaagatgaa gaaaggaatagatcaatggggaatgcagtattagcaggctttctcgttagtggcaagtatgacccagatcttggagattt caaaactattgaagaacttgaaaggctgaaaccgggggaaaaggcaaatatcttactttatcaaggaaagccagttaa agtagttaaaaggaaaaggtatagtgctttgtccaatgacatttcacaaggaattaagagacaaagaatgacagttgagt ccatggggtgggccttgagctaatataaatttatccattaattcaatgaacgcaattgagtg (SEQ ID NO: 162)

Nucleotide atacttttattacaagaaacttccaaactacaataatacaaaaggccagaaacacaatggcagaatttagtgaagatcct sequence of gaattacaaccagcaatgctattcaatatctgcgtccatctagaggtttgctatgtaataagtgacatgaattttcttgacga mouse agaaggaaaagcatatacagcattagaaggacaagggaaagaacaaaatttgagaccacaatatgaagtaattgagg adapted gaatgccaagaaccatagcatggatggtccaaagatccttagctcaagagcatggaatagagactcccaagtatctgg

Influenza ctgatttgtttgattataaaaccaagagatttatagaagttggaataacaaaaggattggctgatgattacttttggaaaaa

B/Malaysia gaaagaaaagttgggaaatagcatggaactgatgatattcagctacaatcaagactactcgttaagtaatgaatcctcat

/2506/2004 tggatgaggaagggaaagggagagtgctaagcagactcacagaacttcaggctgaattaagtctgaaaaacctatgg Sequence Sequence (SEQ ID NO:)

Descriptio

n

virus PA caagttctcataggagaagaagatgttgaaaagggaattgactttaaacttggacaaacaatatctagactaagggatat atctgttcccgctggtttctccaattttgaaggaatgaggagctacatagacaatatagacccaaaaggagcaatagag agaaatctagcaaggatgtctcccttagtatcagtcacacctaaaaagttgacatgggaggacctaagaccaataggg cctcacatttacaaccatgagctaccagaagttccatataatgcctttcttctaatgtctgatgaactggggctggccaata tgactgagggaaagtccaaaaaaccgaagacattagccaaagaatgtctagaaaagtactcaacactacgggatcaa actracccaatattaataatgaaaagcgaaaaagctaatgaaaatttcctatggaagctttggagagactgtgtaaatac aataagtaatgaggaaatgagtaacgagttacagaaaaccaattatgccaagtgggccacaggggatggattaacata ccagaaaataatgaaagaagtagcaatagatgacgaaacaatgtgccaagaagagcctaaaatccctaataaatgta gagtggctgcttgggttcaaacagagatgaatctattgagcactctgacaagtaaaagagctctggacctaccagaaat agggccagacgtagcacccgtggagcatgtagggagtgaaagaaggaaatactttgttaatgaaatcaactactgtaa ggcctctacagttatgatgaagtatgtgctttttcacacttcattgttgaatgaaagcaatgccagcatgggaaaatacaa agtaataccaataaccaatagagtagtaaatgaaacaggagaaagtttcgacatgctttatggtctggcggttaaagga caatctcatctgaggggagatactgatgttgtaacagttgtaactttcgaatttagtagtacagacccaagagtggactca ggaaagtggccaaaatatactgtgtttaggattggctccctatttgtgagtgggagggaaaaatctgtgtacctgtattgc cgagtgaatggcacaaataagatccaaatgaaatggggaatggaagctagaagatgtctgcttcaatcaatgcaacaa atggaagcaattgttgaacaggaatcatcgatacagggatatgacatgaccaaggcttgtttcaagggagacagagta aatagccccaaaactttcagtattggaactcaagaagggaaactagtaaaaggatcctttggaaaagcactaagagta atatttactaaatgtttgatgcactatgtatttggaaatgcccaattggaggggtttagtgccgagtctaggagacttctact gttgatccaagcattaaaggacagaaagggcccttgggtgttcgacttagagggaatgtattctggaatagaagaatgt attagcaacaacccttgggtaatacagagtgcatactggtttaatgaatggttgggctttgaaaaggaggggagtaaag tgttagaatcagtggatgaaataatggatgaataaaaggacatggtactcaatttggtactattttgttcattatgtatctaaa catccaataaaaagaaccaaga (SEQ ID NO: 163)

Nucleotide caacaaaagaactgaaaatcaaaatgtccaacatggatattgacggtatcaacactgggacaattgacaaagcaccg sequence of gaagaaataacttctggaaccagtgggacaaccagaccaatcatcagaccagcaacccttgccccaccaagcaaca mouse aacgaacccggaacccatccccggaaagagcaaccacaatcagtgaagctgatgtcggaaggaaaacccaaaaga adapted aacagaccccgacagagataaagaagagcgtctacaatatggtagtgaaactgggtgaattctataaccagatgatgg Influenza tcaaagctggacttaacgatgacatggagagaaacctaattcaaaatgcgcatgctgtggaaagaattctattggctgc B/Malaysia cactgatgacaagaaaactgaattccagaagaaaaagaatgccagagatgtcaaagaagggaaagaagaaatagat /2506/2004 cacaacaaaacagggggcaccttttacaagatggtaagagatgataaaaccatctacttcagccctataagagtcacct virus NP ttttaaaagaagaagtaaaaacaatgtacaaaaccaccatggggagtgatggcttcagcggactaaatcacataatgat tgggcattcacagatgaatgatgtctgtttccaaagatcaaaggcactaaaaagagttggacttgacccttcattaatcag tacctttgcaggaagcacactccccagaagatcaggtgcaactggtgttgcgatcaaaggaggtggaactctagtggc tgaagccattcgatttataggaagagcaatggcagacagagggctattgagagacatcaaagccaagactgcgtatg aaaagattcttctgaacctaaaaaacaaatgctctgcgccccaacagaaggctctagttgatcaagtgatcggaagtag aaatccagggattgcagacattgaagacctaaccctacttgctcgtagtatggtcgttgttaggccctctgtggcgagca aagtagtgcttcccataagcatttacgccaaaatacctcaactagggttcaacgttgaagagtactctatggttgggtatg aagccatggctctttacaatatggcaacacctgtttccatattaagagtgggagatgatgcaaaggacaaatcacaatta ttcttcatgtcttgcttcggagctgcctatgaagacctgagagttttgtctgcattaacaggcacagaattcaagcctagat cagcattaaaatgcaagggtttccatgttccagcaaaggaacaggtggaaggaatgggggcagctctgatgtccatca agctccagttttgggctccaatgaccagatctgggggaaacgaagtaggtggagacggggggtctggccaaataagt tgcagcccagtgtttgcagtagaaagacctattgctctaagcaagcaagctgtaagaagaatgctgtcaatgaatattga gggacgtgatgcagatgtcaaaggaaatctactcaagatgatgaatgactcaatggctaagaaaaccaatggaaatgc tttcattgggaagaaaatgtttcaaatatcagacaaaaacaaaaccaatcccgttgaaattccaattaagcaaaccatcc Sequence Sequence (SEQ ID NO:)

Descriptio

n

ccaatttcttctttgggagggacacagcagaggattatgatgacctcgattattaaagcaacaaaatagacactatgact gtgattgtttcaatacgtttggaatgtgggtgtttactctta (SEQ ID NO: 164)

Nucleotide aaatgaacaatgctaccttcaactatacaaacgttaaccctatttctcacatcagggggagtattattatcactatatgtgtc sequence of agcttcattgtcatacttactatattcggatatattgctaaaattcccatcaacagaaattactgcaccaacaatgccattgg mouse attgtgcaaacgcatcaaatgttcaggctgtgaaccgttctgcaacaaaaggggtgacacttcttctcccagaaccgga adapted gtggacatacccgcgtttatcttgcccgggctcaacctttcagaaagcactcctaattagccctcatagattcggagaaa Influenza ccaaaggaaactcagctcccttgataataagggaaccttttattgcttgtggaccaaaggaatgcaaacactttgctcta B/Malaysia acccactatgcagcccaaccagggggatactacaatggaacaagaggagacagaaacaagctgaggcatctaattt /2506/2004 cagtcaaattgggcaaaatcccaacagtagaaaactccattttccacatggcagcatggagcgggtccgcatgccatg virus NA atggtaaggaatggacatatatcggagttgatggccctgacaataatgcattgctcaaaataaaatatggagaagcatat actgacacataccattcctatgcaaacaacatcctaagaacacaagaaagtgcctgcaattgcatcgggggaaattgtt atcttatgataactgatggctcagcttcaggtgttagtgaatgcagatttcttaagattcgagagggccgaataataaaag aaatatttccaacaggaagaataaaacatactgaagaatgcacatgcggatttgctagcaataaaaccatagaatgtgc ctgtagagataacagttacacagcaaaaagaccctttgtcaaattaaacgtggagactgatacagcagaaataagattg atgtgcacagagacttatttggacacccccagaccagatgatggaagcataacagggccttgtgaatctaatggggac aaagggagtggaggcatcaagggaggatttgtccatcaaagaatggcatccaagattggaaggtggtactctcgaac gatgtctaaaactaaaaggatggggatggggctgtatgtcaartatgatggagacccatgggctgacagtgatgccctt gcttttagtggagtaatggtttcaatggaagaacctggttggtactcctttggcttcgaaataaaagacaagaaatgtgat gtcccctgtattgggatagagatggtacatgatggtggaaaagagacttggcactcagcagctacagccatttactgttt aatgggctcaggacagctgctgtgggacactgtcacaggtgttaatatggctctgtaatggaggaatggttgagtctgtt ctaaaccctttgttcctattttgtttgaacaattgtccttactgaacttaa (SEQ ID NO: 165)

Nucleotide Cgctgtttggagacacaattgcctacctgctttcattgacagaagatggagaaggcaaagcagaactagcagaaaaat sequence of tacactgttggtttggtgggaaagaatttgacctagactctgccttggaatggataaaaaacaaaagatgcttaactgata mouse tacaaaaagcactaattggtgcctctatatgctttttaaaacccaaagaccaggaaagaaaaagaagattcatcacaga adapted acccttatcaggaatgggaacaacagcaacaaaaaagaaaggcctgattctggctgagagaaaaatgagaagatgt Influenza gtgagctttcatgaagcatttgaaatagcagaaggccatgaaagctcagcgctactatactgtctcatggtcatgtacct B/Malaysia gaatcctggaaattattcaatgcaagtaaaactaggaacgctctgtgctttatgcgagaaacaagcatcacattcacaca /2506/2004 gggctcatagcagagcagcgagatcttcagtgcctggagtgagacgagaaatgcagatggtctcagctatgaacaca vims M gcgaaaacaatgaatggaatgggaaaaggagaagacgtccaaaagctggcagaagagctgcaaagcaacattgga gtgctgagatctcttggggcaagtcaaaagaatggggaaggaattgcaaargatgtaatggaagtgctaaagcagag ctctatgggaaattcagctcttgtgaagaaatatctataatgctcgaaccatttcagattctttcaatttgttcttttatcttatca gctctccatttcatggcttggacaatagggcatttgaatcaaataaaaagaggaataaacatgaaaatacgaataaaag gtccaaacaaagagacaataaacagagaggtatcaattttgagacacagttaccaaaaagaaatccaggccaaagaa acaatgaaggaagtactctctgacaacatggaggtattgagtgaccacataataattgaggggctttctgccgaagaga taataaaaatgggtgaaacagttttggagatagaagaattgcattaaattcaatttttactgtatttcttactatgcatttaagc aaattgtaatcaatgtcagcaaataaactg (SEQ ID NO: 166)

Nucleotide Cgaacaacatgaccacaacacaaattgaggtgggtccgggagcaaccaatgccaccataaactttgaagcaggaat sequence of tctggagtgctatgaaaggctttcatggcaaagagcccttgactaccctggtcaagaccgcctaaacagactaaagag mouse aaaattagagtcaagaataaagactcacaacaaaagtgagcctgaaagtaaaaggatgtcccttgaagagagaaaag adapted caattggagtaaaaatgatgaaagtactcctatttatggatccgtctgctggaattgaagggtttgagccatactgtatga

Influenza aaagttcctcaaatagcaactgtacgaaatacaattggaccgattacccttcaacaccagggaggtgccttgatgacat

B/Malaysia agaagaagaaccagaggatgttgatggcccaactgaaatagtattaagggacatgaacaacaaagatgcaaggcaa

/2506/2004 aagataaaggaggaagtaaacactcagaaagaagggaagttccgtttgacaataaaaagggatatgcgtaatgtattg Sequence Sequence (SEQ ID NO:)

Descriptio

n

virus NS tccttgagagtgttggtaaacggaacattcctcaaacatcccaatggatacaagtccttatcaactctgcatagattgaat gcatatgaccagagtggaaggcttgttgctaaacttgttgctactgatgatcttacagtggaggatgaagaagatggcc atcggatcctcaactcactcttcgagcgtcttaatgaaggacattcaaagccaattcgagcagctgaaactgcggtggg agtcttatcccaatttggtcaagagcaccgattatcaccagaagagggagacaattagactggtcacggaagaacttta tcttttaagtaaaagaattgatgataacatattgttccacaaaacagtaatagctaacagctccataatagctgacatggtt gtatcattatcattattagaaacattgtatgaaatgaaggatgtagttgaagtgtacagcaggcagtgcttgtgaatttaaa ataaaa (SEQ ID NO: 167)

[00678] To generate the mH13/B chimeric HA for rescue in an influenza B/Malaysia/2506/04 MA virus backbone, amino acid residues relating to the 120 loop, 150 loop, 160 loop, and 190 helix of the influenza B/Yamagata/16/88 virus HA globular head were modified based on amino acids from the influenza A/black headed gull/Sweden/1/99 virus globular head domain (FIGS. 34 and 35). In particular, the amino acid sequences of the influenza B/Yamagata/16/88 virus HA 120 loop (TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO: 108)), 150 loop

(PNVTSRNG (SEQ ID NO: 123)), 160 loop (RDNKTA (SEQ ID NO: 110)), and 190 helix (NKNQMKN (SEQ ID NO: 112)) were replaced with amino acid sequences NIP,

RIELSTHNVINAEVAPGGPYRL (SEQ ID NO: 168), PDKGASS (SEQ ID NO: 133),

KRGNQY (SEQ ID NO: 134), and VSTNMAK (SEQ ID NO: 135), respectively, based on amino acid sequences of the influenza A/black headed gull/Sweden/1/99 virus globular head domain. Influenza B/Malaysia/2506/04 MA virus was used as the backbone (i.e., the PB2, PB1, PA, NP, NA, M, and NS are from influenza B/Malaysia/2506/04 MA virus) to rescue virus expressing this chimeric HA construct. Additionally, the glutamic acid (E) amino acid at position 156 of the immature influenza B/Yamagata/16/88 virus HA was substituted with a lysine (K) (i.e., an E156K mutation) in the chimeric HA. This virus was successfully rescued, having a stock titer of 2.19 x 10⁸ PFU/mL. This virus was slightly attenuated when grown in 10- day old embryonated eggs (500 PFU/egg inoculant) at 33 degrees Celsius, as compared to a control influenza B/Malaysia/2506/04 MA virus (FIG. 36D). The peak titer p value was 0.0072.

[00679] The ability of these chimeric HAs to bind cross-protective antibodies that bind the influenza virus stalk and/or head domains was evaluated. To this end, antibodies CR9114, 5A7, CR8059, CR8033, and Antibody X (sometimes referred to as IIC7) were utilized. A summary of these antibodies is provided in Table 34, below. The tested chimeric HA polypeptides retained their abilities to be recognized by the tested anti-stalk and anti-head antibodies (FIGS. 37A- 37D). Surface staining was performed on MDCK cells infected with the indicated viruses at a multiplicity of infection of 5 without TPCK-trypsin at 17 hours post-infection with monoclonal antibodies known to bind influenza B HA head or stalk domains (FIGS. 37A-37D).

[00680] Table 34. Cross-protective antibodies utilized.

[00681] The HI activity of the chimeric HAs was tested to determine whether the antigenic loops of the influenza B virus were successfully replaced. Specifically, HI assays were performed with mouse or ferret serum raised against wild type influenza B/Yamagata/16/88 virus (FIGS. 38A-38D). The chimeric HA polypeptides displayed drastically reduced strain specific HI activity (FIGS. 38A-38D). This was an indirect method for evaluating that antigenic sites within the influenza B virus HA had either been replaced, or in the least ablated, by swapping in exotic influenza A virus HA sequences (such as those from H5, H8, HI 1, or HI 3).

[00682] In conclusion, chimeric HA constructs can be incorporated into rescued influenza B viruses that grow to robust titers in eggs, despite being attenuated compared to the wild type virus. The chimeric HAs appeared to lose their B HA immunodominant head epitopes, while sub-dominant conserved, cross-protective epitopes on B HA were preserved. EXAMPLE 8: VACCINATION REGIMENS COMPRISING CHIMERIC HA

[00683] Generation of Chimeric HA Constructs: mH5/B, mH8/B, and mH13/B chimeric HA constructs were generated as described in Section 6.6. pDZ plasmid (see Martinez-Sobrido L, Garcia-Sastre A. Generation of Recombinant Influenza Virus from Plasmid DNA. Journal of Visualized Experiments : JoVE. 2010;(42):2057. doi: 10.3791/2057, which is incorporated by reference herein in its entirety) encoding the mH13/B chimeric HA was giga-prepped (service provided by Genewiz) to prepare the DNA used for the prime (see Table 35, infra). mH5/B and mH8/B chimeric HA recombinant proteins were synthesized using baculovirus expression system as previously described in Margine I, Palese P, Krammer F. Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System. Journal of Visualized Experiments : JoVE.

2013;(81):51112. doi: 10.3791/51112, which is incorporated by reference herein in its entirety.

[00684] Mouse Models and In Vivo Analyses: Five 6 to 8 weeks old female BALB/c mice/group (ten groups in total) were used in this example (see Table 35, below). Mice are vaccinated as indicated in Table 35, below. Specifically, mice are primed with either: (i) 80 μg of pDZ plasmid encoding mH13/B administered by electroporation (Groups 1, 2, 5-7, and 10); (ii) 1 μg of Fluzone (2006-2007 season, comprising an influenza B virus of the Victoria lineage; Sanofi Pasteur) administered intramuscularly (Group 3); (iii) 1 μg of Flulaval (2008-2009 season, comprising an influenza B virus of the Yamagata lineage; GlaxoSmithKline)

administered intramuscularly (Group 8); or (iv) mock primed (Groups 4 and 9). Three weeks post-prime, mice in Groups 1, 2, 4-7, 9, and 10 are boosted with: (i) intramuscular administration of 5 μg of mH5/B protein adjuvanted with 5 μg of polyLC and intranasal administration of 5 μg of mH5/B protein adjuvanted with 5 μg of polyLC (Groups 1, 5, 6, and 10); or (ii) intramuscular administration of 5 μg of bovine serum albumin (BSA) adjuvanted with 5 μg of polyLC and intranasal administration of 5 μg of BSA adjuvanted with 5 μg of polyLC (Groups 2, 4, 7, and 9). Mice in Groups 3 and 8 do not receive a boost three-weeks post-prime. Six weeks post- prime, mice are boosted with: (i) intramuscular administration of 5 μg of mH8/B protein adjuvanted with 5 μg of polyLC and intranasal administration of 5 μg of mH8/B protein adjuvanted with 5 μg of polyLC (Groups 1, 5, 6, and 10); (ii) intramuscular administration of 5 μg of BSA adjuvanted with 5 μg of polyLC and intranasal administration of 5 μg of BSA adjuvanted with 5 μg of polyLC (Groups 2, 4, 7, and 9); (iii) 1 μg of Fluzone administered intramuscularly (Group 3); or (iv) 1 μ of Flulaval administered intramuscularly (Group 8). Four weeks later (i.e., 10 weeks after the prime), mice in Groups 1-4 were challenged with influenza B/Malaysia/2506/04 virus (Victoria lineage) at 5 mouse lethal dose 50 ("LD50"), intranasally, and mice in Groups 1-4 were monitored for weight loss (FIG. 39 A) and survival (FIG. 59B) compared to naive mice. Mice in Group 1 were completely protected from mortality with minimal weight loss.

[00685] Additionally, 10 weeks after the prime, mice in Groups 6-9 are challenged with influenza B/Florida/4/06 virus (Yamagata lineage) at 5 mouse LD50, intranasally, and mice in Groups 5 and 10 are terminally bled (for, e.g., passive transfer studies). The challenged mice are monitored for weight loss and survival.

[00686] Table 35. Chimeric HA vaccination in mice. TIV=trivalent influenza vaccine;

BSA=bovine serum albumin; FM=intramuscular; IN=intranasally; VIC indicates that the virus is from the influenza B virus Victoria lineage; YAM indicates that the virus is from the influenza B virus Yamagata lineage. mH5/B refers to mH5/B chimeric HA described in FIGS. 28 and 29. mH8/B refers to the mH8/B chimeric HA described in FIGS. 30 and 31. mH13/B refers to the mH13/B chimeric HA described in FIGS. 32 and 33.

80 μg 10 μ polyLC 10 μ polyLC

7. EMBODIMENTS

[00687] Provided herein are the following exemplary embodiments:

1. An immunogenic composition comprising a chimeric influenza virus hemagglutinin (HA) polypeptide and a liposomal adjuvant, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

2. An immunogenic composition comprising an inactivated influenza virus and a liposomal adjuvant, wherein the inactivated influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

3. An immunogenic composition comprising a split influenza virus and a liposomal adjuvant, wherein the split influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

4. The immunogenic composition of embodiment 1 which is a subunit vaccine.

5. The immunogenic composition of any one of embodiments 1 to 4, wherein the influenza virus HA globular head domain is from one influenza A virus subtype and the HA stem domain polypeptide is from a different influenza A virus subtype.

6. The immunogenic composition of embodiment or 45, wherein the HA stem domain is from influenza A virus subtype HI .

7. The immunogenic composition of embodiment or 45, wherein the HA stem domain is from influenza A virus subtype H3.

8. The immunogenic composition of any one of embodiments 4 to 18, wherein the influenza virus HA globular head domain is from influenza A virus H8.

9. The immunogenic composition of any one of embodiments 4 to 18, wherein the influenza virus HA globular head domain is from influenza A virus H5.

10. The immunogenic composition of any one of embodiments 4 to 18, wherein the influenza virus HA globular head domain is from influenza A virus subtype H4, H6, H7, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18.

11. The immunogenic composition of any one of embodiments 1 to 4, wherein the influenza virus HA globular head domain is from one influenza virus species and the HA stem domain is from a different influenza virus species. 12. The immunogenic composition of any one of embodiments 1 to 4, wherein the influenza virus HA globular head domain is from an influenza A virus and the HA stem domain polypeptide is from an influenza B virus lineage.

13. The immunogenic composition of embodiments 1 to 4 which further comprises an influenza virus neuraminidase (NA) polypeptide.

14. The immunogenic composition of embodiment 2, wherein the inactivated influenza virus comprises an influenza virus NA polypeptide.

15. The immunogenic composition of embodiment 3, wherein the split influenza virus comprises an influenza virus NA polypeptide.

16. The immunogenic composition of any one of embodiments 4 to 19 which comprises an influenza virus NA polypeptide that is from the influenza virus from which the HA stem domain polypeptide is obtained.

17. The immunogenic composition of embodiment 20 which comprises an influenza virus NA polypeptide that is from the influenza virus from which the HA stem domain polypeptide is obtained.

18. The immunogenic composition of embodiment 21 which comprises an influenza virus NA polypeptide that is from the influenza virus from which the HA stem domain polypeptide is obtained.

19. The immunogenic composition of any one of embodiments 1 to 11 or 13 to 15, wherein the HA stem domain polypeptide 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 HAlN-term through Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HA1 domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

20. The immunogenic composition of any one of embodiments 1 to 11 or 13 to 15, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

21. The immunogenic composition of any one of embodiments 1 to 11 or 13 to 15, wherein 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 HAlN-term through Ap of an influenza A virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq.

22. The immunogenic composition of any one of embodiments 1 to 4, 11 to 15, 17 or 18, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering.

23. The immunogenic composition of any one of embodiments 1 to 4, 11 to 15, 17 or 18, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

24. The immunogenic composition of any one of embodiments 1 to 4, 11 to 15, 17 or 18, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering. 25. The immunogenic composition of any one of embodiments 1 to 4, 11 to 15, 17 or 18, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N-terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq in the HAl C-terminal stem segment is substituted with a Cys.

26. The immunogenic composition of any one of embodiments 1 to 4, 11 to 15, 17 or 18, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

27. The immunogenic composition of any one of embodiments 1 to 4, 11 to 15, 17 or 18, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N-terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dq in the HAl C-terminal stem segment is substituted with a Cys, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

28. The immunogenic composition of any one of embodiments 1 to 27, wherein the influenza virus HA stem domain further comprises the stem domain of an influenza virus HA2 domain.

29. The immunogenic composition of any one of embodiments 1 to 28, wherein the influenza virus HA stem domain further comprises an influenza virus HA2 domain lacking the cytoplasmic domain and transmembrane domain.

30. The immunogenic composition of embodiment 29, wherein the HA2 domain is from the same strain as the N-terminal stem segment.

31. The immunogenic composition of embodiment 29 or 30, wherein the HA2 domain is from the same strain as the C-terminal stem segment.

32. The immunogenic composition of any one of embodiments 1 to 3, wherein the HA stem domain polypeptide is from an influenza B virus HA, and wherein the HA globular head domain is the globular head domain derived from the influenza B virus HA and comprises one, two, three or all of the following:

a. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; b. 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA;

c. 2, 3, 4, 5 or more amino acid substitutions within the 160 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; and d. 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA.

33. The immunogenic composition of embodiment 32, wherein the chimeric influenza virus HA polypeptide further comprises the transmembrane domain and cytoplasmic tail domain of the influenza B virus HA.

34. The immunogenic composition of embodiment 32 or 33, wherein the influenza B virus is of the Yamagata lineage or of the Victoria lineage.

35. The immunogenic composition of embodiment 32 or 33, wherein the influenza B virus is influenza B/Yamagata/16/88.

36. The immunogenic composition of any one of embodiments 32 to 35, wherein the influenza A virus is an influenza A virus of an H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18. 37. The immunogenic composition of embodiment 35, wherein the influenza A virus is an H5 HA subtype.

38. The immunogenic composition of embodiment 37, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues FIP and KIQL STKNVINAEH APGGP YRL (SEQ ID NO:

109) ; b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PYQGKSS (SEQ ID NO: 124); c. the acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KKNSTY (SEQ ID NO: 111); and/or d. the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NDAAMQT (SEQ ID NO: 113).

39. The immunogenic composition of embodiment 37 or 38, wherein the H5 subtype is influenza A/Vietnam/1203/04 (HALo) virus.

40. The immunogenic composition of embodiment 35, wherein the influenza A virus is an H8 HA subtype.

41. The immunogenic composition of embodiment 40, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues HIP and RIRLSTYNVINAETAPGGPYRL (SEQ ID NO:

125); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NASTGGQS (SEQ ID NO: 126); c. the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KKKADTY (SEQ ID NO: 127); and/or d. the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues ADAKMQT (SEQ ID NO: 128).

42. The immunogenic composition of embodiment 40 or 41, wherein the H8 subtype is influenza A/Mallard/Sweden/24/2002 virus.

43. The immunogenic composition of embodiment 35, wherein the influenza A virus is an HI 1 HA subtype.

44. The immunogenic composition of embodiment 43, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LIP and KIEL S TSN VIN AEV APGGP YRL (SEQ ID NO: 129); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PFGSSNS (SEQ ID NO: 130); c. the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues HQSGTY (SEQ ID NO: 131); and/or d. the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues TTLKMHQ (SEQ ID NO: 132).

45. The immunogenic composition of embodiment 43 or 44, wherein the HI 1 subtype is influenza A/northern shovel er/Netherlands/18/99 virus.

46. The immunogenic composition of embodiment 35, wherein the influenza A virus is an HI 2 HA subtype.

47. The immunogenic composition of embodiment 46, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues YIP and RIKLSTFN VIN AET APGGP YRL (SEQ ID NO: 136); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NNTSNQGS (SEQ ID NO: 137); c. the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LKSGQF (SEQ ID NO: 138); and/or d. the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PTSDMQI (SEQ ID NO: 139). 48. The immunogenic composition of embodiment 46 or 47, wherein the H12 subtype is influenza A/mallard/interior Alaska/7MP0167/2007 virus.

49. The immunogenic composition of embodiment 35, wherein the influenza A virus is an HI 3 HA subtype.

50. The immunogenic composition of embodiment 49, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NIP and RIELSTHNVINAEV APGGP YRL (SEQ ID NO: 168); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PDKGASS (SEQ ID NO: 133); c. the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues KRGNQY (SEQ ID NO: 134); and/or d. the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues VSTNMAK (SEQ ID NO: 135).

51. The immunogenic composition of embodiment 49 or 50, wherein the H13 subtype is influenza A/black headed gull/Sweden/1/99 virus.

52. The immunogenic composition of any one of embodiments 32 to 51, wherein the HA globular head domain comprises one, two, or more amino acid substitutions outside of one, two, three, or all of the following: the 120 loop, the 150 loop, the 160 loop, and the 190 helix. 53. The immunogenic composition of any one of embodiments 1 to 52, wherein the liposomal adjuvant comprises a TLR4 agonist and saponin.

54. The immunogenic composition of any one of embodiments 1 to 53, wherein the TLR 4 agonist is a lipopolysaccharide.

55. The immunogenic composition of any one of embodiments 1 to 53, wherein the TLR 4 agonist is a non-toxic derivative of lipid A.

56. The immunogenic composition of any one of embodiments 1 to 53, wherein the TLR 4 agonist is a monophosphoryl lipid A.

57. The immunogenic composition of any one of embodiments 1 to 53, wherein the TLR 4 agonist is 3-de-O-acylated monophosphoryl lipid A.

58. The immunogenic composition of any one of embodiments 1 to 53, wherein the saponin is obtainable from Quillaja saponaria.

59. The immunogenic composition of any one of embodiments 1 to 58, wherein the saponin comprises one or more of QS7, QS17, QS18 and QS21.

60. The immunogenic composition of any one of embodiments 1 to 58, wherein the saponin comprises QS21.

61. The immunogenic composition of any one of embodiments 1 to 52, wherein the TLR4 agonist is 3-O-desacyl-monophosphoiyl lipid A and the saponin is QS21.

62. The immunogenic composition of any one of embodiments 1 to 61, wherein the liposomes are 30-200 nm in size. 63. The immunogenic composition of any one of embodiments 1 to 61, wherein the liposomes are 95-120 nm in size.

64. The immunogenic composition of any one of embodiments 1 to 61, wherein the liposomes comprise l,2-dioleoyl-sn-glycero-3 -phosphatidylcholine (DOPC).

65. The immunogenic composition of any one of embodiments 1 to 64, wherein the composition comprises a human dose volume of 0.05 ml to 1 ml of the lipososomal adjuvant.

66. The immunogenic composition of any one of embodiments 1 to 64, wherein the composition comprises a human dose volume of 0.4 ml to 0.6 ml of the lipososomal adjuvant.

67. The immunogenic composition of any one of embodiments 1 to 64, wherein the lipososomal adjuvant comprises 1-100 μg of the TLR4 agonist per human dose.

68. The immunogenic composition of any one of embodiments 1 to 64, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose.

69. The immunogenic composition of any one of embodiments 1 to 64, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist per human dose.

70. The immunogenic composition of any one of embodiments 1 to 64, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose.

71. The immunogenic composition of any one of embodiments 1 to 64, wherein the lipososomal adjuvant comprises 1-100 μg of the saponin per human dose.

72. The immunogenic composition of any one of embodiments 1 to 64, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist and 20-30 μg of saponin per human dose. 73. The immunogenic composition of any one of embodiments embodiments 1 to 64, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist and 40-60 μg of saponin per human dose.

74. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising administering to the subject the immunogenic composition of any one of

embodiments 1 to 73.

75. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition comprising a live attenuated influenza virus comprising a first chimeric hemagglutinin (HA) polypeptide, wherein the first chimeric HA polypeptide 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 immunogenic composition, administering to the subject a second immunogenic composition which is an immunogenic composition according to any one of embodiments 1 to 73, wherein the chimeric influenza virus HA of the second immunogenic composition comprises an influenza virus HA globular head domain that is different than the influenza virus HA globular head domain of the first chimeric HA polypeptide, and wherein the chimeric influenza virus HA of the second immunogenic composition comprises the same HA stem domain polypeptide as the first chimeric HA polypeptide.

76. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition which is the immunogenic composition of any one of embodiments 1 to 73; and

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the second chimeric influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a second influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is heterologous to the second HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is different than the influenza virus HA globular head domain of the chimeric influenza virus HA polypeptide of the first immunogenic composition, and wherein the second HA stem domain polypeptide is the same as the HA stem domain polypeptide of the chimeric influenza virus HA polypeptide of the first immunogenic composition.

77. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises an inactivated influenza virus comprising a second chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the second chimeric influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a second influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is heterologous to the second HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is different than the influenza virus HA globular head domain of the chimeric influenza virus HA polypeptide of the first immunogenic composition, and wherein the second HA stem domain polypeptide is the same as the HA stem domain polypeptide of the chimeric influenza virus HA polypeptide of the first immunogenic composition.

78. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition which is the immunogenic composition of any one of embodiments 1 to 73; and (b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a split influenza virus and the liposomal adjuvant, wherein the split influenza virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a second influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is

heterologous to the second HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is different than the influenza virus HA globular head domain of the chimeric influenza virus HA polypeptide of the first immunogenic composition, and wherein the second HA stem domain polypeptide is the same as the HA stem domain polypeptide of the chimeric influenza virus HA polypeptide of the first immunogenic composition.

79. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition comprising a first chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype.

80. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition comprising a first inactivated influenza A virus and a liposomal adjuvant, wherein the first inactivated influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second inactivated influenza A virus and the liposomal adjuvant, wherein the second inactivated influenza A virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype.

81. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition comprising a first split influenza A virus and a liposomal adjuvant, wherein the first split influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

(b) a certain time after the administration of first the immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second split influenza A virus and the liposomal adjuvant, wherein the second split influenza A virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype.

82. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition comprising a first chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype.

83. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition comprising a first inactivated influenza A virus and a liposomal adjuvant, wherein the first inactivated influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second inactivated influenza A virus and the liposomal adjuvant, wherein the second inactivated influenza A virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype.

84. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject a first immunogenic composition comprising a first split influenza A virus and a liposomal adjuvant, wherein the first split influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second split influenza A virus and the liposomal adjuvant, wherein the second split influenza A virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype.

85. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising administering to the subject a second immunogenic composition which is an immunogenic composition according to any one of embodiments 1 to 73 a certain period of time after the administration of a first immunogenic composition, wherein the first immunogenic composition comprises a live attenuated influenza virus comprising a first chimeric

hemagglutinin (HA) polypeptide, wherein the first chimeric HA polypeptide 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, wherein the chimeric influenza virus HA of the second immunogenic composition comprises an influenza virus HA globular head domain that is different than the influenza virus HA globular head domain of the first chimeric HA polypeptide, and wherein the chimeric influenza virus HA of the second immunogenic composition comprises the same HA stem domain polypeptide as the first chimeric HA polypeptide.

86. A method for immunizing against influenza virus in a subject (e.g., a human subject), comprising administering to the subject a second immunogenic composition a certain period of time after the administration of a first immunogenic composition to the subject, wherein the first immunogenic composition is the immunogenic composition of any one of embodiments 1 to 73, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the second chimeric influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a second influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is heterologous to the second HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is different than the influenza virus HA globular head domain of the chimeric influenza virus HA polypeptide of the first immunogenic composition, wherein the second HA stem domain polypeptide is the same as the HA stem domain polypeptide of the chimeric influenza virus HA polypeptide of the first immunogenic composition.

87. A composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising administering the subject the immunogenic composition according to any one of embodiments 1 to 73.

88. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the first immunogenic composition, wherein the first immunogenic composition comprises a live attenuated influenza virus comprising a first chimeric hemagglutinin (HA) polypeptide, wherein the first chimeric HA polypeptide 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

89. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the first immunogenic composition which is the immunogenic composition of any one of embodiments 1 to 73; and

90. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

91. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a split influenza virus and the liposomal adjuvant, wherein the split influenza virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a second influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is

92. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the first immunogenic composition, wherein the first immunogenic composition comprises a first chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

93. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the first immunogenic composition, wherein the first immunogenic composition comprises a first inactivated influenza A virus and a liposomal adjuvant, wherein the first inactivated influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA

polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

94. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the first immunogenic composition, wherein the first immunogenic composition comprises a first split influenza A virus and a liposomal adjuvant, wherein the first split influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

95. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the first immunogenic composition, wherein the first immunogenic composition comprises a first chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

96. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

97. A first immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the first immunogenic composition, wherein the first immunogenic composition comprises a first split influenza A virus and a liposomal adjuvant, wherein the first split influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and

98. A second immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising administering to the subject the second immunogenic composition which is an immunogenic composition according to any one of embodiments 1 to 73 a certain period of time after the administration of a first immunogenic composition, wherein the first immunogenic composition comprises a live attenuated influenza virus comprising a first chimeric hemagglutinin (HA) polypeptide, wherein the first chimeric HA polypeptide 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, wherein the chimeric influenza virus HA of the second immunogenic composition comprises an influenza virus HA globular head domain that is different than the influenza virus HA globular head domain of the first chimeric HA polypeptide, and wherein the chimeric influenza virus HA of the second immunogenic composition comprises the same HA stem domain polypeptide as the first chimeric HA polypeptide.

99. A second immunogenic composition for use in a method for immunizing against influenza virus in a subject (e.g., a human subject), the method comprising administering to the subject the second immunogenic composition a certain period of time after the administration of a first immunogenic composition to the subject, wherein the first immunogenic composition is the immunogenic composition of any one of embodiments 1 to 73, wherein the second immunogenic composition comprises a second chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the second chimeric influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and a second influenza virus HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is heterologous to the second HA stem domain polypeptide, wherein the second influenza virus HA globular head domain is different than the influenza virus HA globular head domain of the chimeric influenza virus HA polypeptide of the first immunogenic composition, wherein the second HA stem domain polypeptide is the same as the HA stem domain polypeptide of the chimeric influenza virus HA polypeptide of the first immunogenic composition, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

100. The method of embodiment 79 or 82, or the first immunogenic composition for the use of embodiment 92 or 95, wherein the first and second immunogenic compositions are subunit vaccines.

101. The method of embodiment 79 or 82, or the first immunogenic composition for the use of embodiment 92 or 95 further comprising administering a third immunogenic composition, wherein the third immunogenic composition comprises a third chimeric influenza virus HA polypeptide and the liposomal adjuvant , wherein the third chimeric influenza virus HA polypeptide comprises a third influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the third influenza virus HA globular head domain is the HA globular head domain of an influenza A virus HI 1 subtype.

102. The method of embodiment 80 or 83, or the first immunogenic composition for the use of embodiment 93 or 96 further comprising administering a third immunogenic composition, wherein the third immunogenic composition comprises a third inactivated influenza A virus, wherein the third inactivated influenza A virus comprises a third chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the third chimeric influenza virus HA polypeptide comprises a third influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the third influenza virus HA globular head domain is the HA globular head domain of an influenza A virus HI 1 subtype.

103. The method of embodiment 81 or 84, or the first immunogenic composition for the use of embodiment 94 or 97 further comprising administering a third immunogenic composition, wherein the third immunogenic composition comprises a third split influenza A virus, wherein the third split influenza A virus comprises a third chimeric influenza virus HA polypeptide and the liposomal adjuvant, wherein the third chimeric influenza virus HA polypeptide comprises a third influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the third influenza virus HA globular head domain is the HA globular head domain of an influenza A virus HI 1 subtype.

104. The method or the first immunogenic composition for the use of embodiment 102, wherein the first, second and third inactivated influenza A viruses comprise an influenza A virus neuraminidase from the same influenza virus strain as the influenza virus strain of the first HA stem domain polypeptide.

105. The method or the first immunogenic composition for the use of embodiment 103, wherein the first, second and third split influenza A viruses comprise an influenza A virus neuraminidase from the same influenza virus strain as the influenza virus strain of the first HA stem domain polypeptide.

106. The method or the first immunogenic composition for the use of any one of embodiments 101 to 103, wherein the influenza A virus HI 1 subtype is A/Northern

Shoveloer/Netherlands/18/1999.

107. The method of embodiment 80 or 83, or the first immunogenic composition for the use of embodiment 93 or 96, wherein the first and second inactivated influenza A viruses comprise an influenza A virus neuraminidase from the same influenza virus strain as the influenza virus strain of the first HA stem domain polypeptide.

108. The method of embodiment 81 or 84, or the first immunogenic composition for the use of embodiment 94 or 97, wherein the first and second split influenza A viruses comprise an influenza A virus neuraminidase from the same influenza virus strain as the influenza virus strain of the first HA stem domain polypeptide.

109. The method of any one of embodiments 79 to 84 or 100 to 108, or the first immunogenic composition for the use of any one of embodiments 92 to 108, wherein the first and second HA stem domain polypeptides comprise 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 Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Aq through HA C- term of an influenza A virus hemagglutinin HA1 domain, wherein HAN-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 HA1 domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein the first and second influenza A virus HA globular head domains consist of the amino acid residues intervening Ap and Aq. 110. The method or the first immunogenic composition for the use of any one of embodiments 101 to 106, wherein the first, second and third HA stem domain polypeptides comprise 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 Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Aq through HA C-term of an influenza A virus hemagglutinin HA1 domain, wherein HAN-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 HA1 domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein the first, second and third influenza A virus HA globular head domains consist of the amino acid residues intervening Ap and Aq.

111. The method of any one of embodiments 79 to 84 or 110, or the first immunogenic composition for the use of any one of embodiments 92 to 97 or 1 10 to 112, wherein the influenza A virus H5 subtype is influenza virus A/Vietnam/1203/2004.

112. The method of any one of embodiments 79 to 84, 110 or 111, or the first immunogenic composition for the use of any one of embodiments 92 to 97, 110 or 111, wherein the influenza A virus H8 subtype is influenza virus A/mallard/Sweden/24/2002.

113. The method of any one of embodiments 79 to 84 or 110 to 112, or the first immunogenic composition for the use of any one of embodiments 92 to 97 or 110 to 112, wherein the influenza A virus HI subtype is A/California/04/2009.

114. The method of any one of embodiments 75 to 86 or 100 to 113 or the

immunogenic composition for the use of any one of embodiments 88 to 97 or 100 to 113, wherein the second immunogenic composition is administered 1 week to 9 months, 3 weeks to 8 months, 6 weeks to 12 weeks, 4 weeks to 6 months, 5 weeks to 5 months, 6 weeks to 4 months, 7 weeks to 4 months, 8 weeks to 4 months, 8 weeks to 3 months, 3 months to 6 months, 3 months to 9 months, or 6 months to 9 months after the administration of the first immunogenic composition.

115. A method for immunizing against an influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject an immunogenic composition comprising a chimeric

influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and

(b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant

comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

116. A method for immunizing against an influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject an immunogenic composition comprising an inactivated influenza virus, wherein the inactivated influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and

117. A method of immunizing against influenza virus in a subject (e.g., a human subject), comprising:

(a) administering to the subject an immunogenic composition comprising a split influenza virus and a liposomal adjuvant, wherein the split influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and

118. An immunogenic composition for use in a method for immunizing against an influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises a chimeric influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and

119. An immunogenic composition for use in a method for immunizing against an influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises an inactivated influenza virus, wherein the inactivated influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and

120. An immunogenic composition for use in a method of immunizing against influenza virus in a subject (e.g., a human subject), the method comprising:

(a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises a split influenza virus and a liposomal adjuvant, wherein the split influenza vims comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant

121. The method of embodiment 115 or the immunogenic composition for the use of embodiment 111 which is a subunit vaccine.

122. The method of any one of embodiments 115 to 117 or 121, or the immunogenic composition for the use of any one of embodiments 118 to 121, wherein the influenza virus HA globular head domain is from one influenza A virus subtype and the HA stem domain

polypeptide is from a different influenza A virus subtype.

123. The method or the immunogenic composition for the use of embodiment 122, wherein the HA stem domain is from influenza A virus subtype HI .

124. The method or the immunogenic composition for the use of embodiment 122, wherein the HA stem domain is from influenza A virus subtype H3.

125. The method or the immunogenic composition for the use of any one of embodiments 122 to 124, wherein the influenza virus HA globular head domain is from influenza A virus H8.

126. The method or the immunogenic composition of any one of embodiments 122 to 124, wherein the influenza virus HA globular head domain is from influenza A virus H5.

127. The method or the immunogenic composition of any one of embodiments 122 to 124, wherein the influenza virus HA globular head domain is from influenza A virus subtype H4, H6, H7, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18. 128. The method of any one of embodiments 115 to 117 or 121, or the immunogenic composition for the use of any one of embodiments 118 to 121, wherein the influenza virus HA globular head domain is from one influenza virus species and the HA stem domain is from a different influenza virus species.

129. The method of any one of embodiments 115 to 117 or 121, or the immunogenic composition for the use of any one of embodiments 118 to 121, wherein the influenza virus HA globular head domain is from an influenza A virus and the HA stem domain polypeptide is from an influenza B virus lineage.

130. The method of embodiment 115 or 121, or the immunogenic composition for the use of embodiment 111 or 114 which further comprises an influenza virus neuraminidase (NA) polypeptide.

131. The method of embodiment 116, or the immunogenic composition for the use of embodiment 119, wherein the inactivated influenza virus comprises an influenza virus NA polypeptide.

132. The method of embodiment 116, or the immunogenic composition for the use of embodiment 120, wherein the split influenza virus comprises an influenza virus NA polypeptide.

133. The method or the immunogenic composition for the use of any one of embodiments 122 to 127 which comprises an influenza virus NA polypeptide that is from the influenza virus from which the HA stem domain polypeptide is obtained.

134. The method or the immunogenic composition for the use of embodiment 128 which comprises an influenza virus NA polypeptide that is from the influenza virus from which the HA stem domain polypeptide is obtained. 135. The method or the immunogenic composition for the use of embodiment 129 which comprises an influenza virus NA polypeptide that is from the influenza virus from which the HA stem domain polypeptide is obtained.

136. The method of any one of embodiments 115 to 117, 121 to 128 or 130 to 134, or the immunogenic composition for the use of any one of embodiments 118 to 128 to 130 to 134, wherein the HA stem domain polypeptide 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 HAlN-term through Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HA1 domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HA1 domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

137. The method of any one of embodiments 115 to 117, 121 to 128 or 130 to 134, or the immunogenic composition for the use of any one of embodiments 118 to 128 to 130 to 134, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

138. The embodiments 115 to 117, 121 to 128 or 130 to 134, or the immunogenic composition for the use of any one of embodiments 118 to 128 to 130 to 134, wherein the HA stem domain polypeptide 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 HAlN- term through Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HA1 C- terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein the influenza A virus HA globular head domain consists of the amino acid residues intervening Ap and Aq.

139. The method of any one of embodiments 115 to 117, 121, 129 or 135, or the immunogenic composition for the use of any one of embodiments 118 to 121, 129 or 135, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering.

140. The method of any one of embodiments 115 to 117, 121, 129 or 135, or the immunogenic composition for the use of any one of embodiments 118 to 121, 129 or 135, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

141. The method of any one of embodiments 115 to 117, 121, 129 or 135, or the immunogenic composition for the use of any one of embodiments 118 to 121, 129 or 135, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

142. The method of any one of embodiments 115 to 117, 121, 129 or 135, or the immunogenic composition for the use of any one of embodiments 118 to 121, 129 or 135, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N-terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq in the HAl C-terminal stem segment is substituted with a Cys. 143. The method of any one of embodiments 115 to 117, 121, 129 or 135, or the immunogenic composition for the use of any one of embodiments 118 to 121, 129 or 135, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

144. The method of any one of embodiments 115 to 117, 121, 129 or 135, or the immunogenic composition for the use of any one of embodiments 118 to 121, 129 or 135, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N-terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dq in the HAl C-terminal stem segment is substituted with a Cys, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

145. The method of any one of embodiments 115 to 117 or 121, or the immunogenic composition for the use of any one of embodiments 117 to 120, wherein the HA stem domain polypeptide is from an influenza B virus HA, and wherein the HA globular head domain is the globular head domain derived from the influenza B virus HA and comprises one, two, three or all of the following:

c. 2, 3, 4, 5 or more amino acid substitutions within the 160 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5 or more amino acid residues in the 160 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; and d. 2, 3, 4, 5, 6, 7, 8 or more amino acid substitutions within the 190 helix of the

globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8 or more amino acid residues in the 190 helix of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA.

146. The method or the immunogenic composition for the use of embodiment 145, wherein the chimeric influenza virus HA polypeptide further comprises the transmembrane domain and cytoplasmic tail domain of the influenza B virus HA. 147. The method or the immunogenic composition for the use of embodiment 145 or 146, wherein the influenza B virus is of the Yamagata lineage or of the Victoria lineage.

148. The method or the immunogenic composition for the use of embodiment 145 or 146, wherein the influenza B virus is influenza B/Yamagata/16/88.

149. The method or the immunogenic composition for the use of any one of

embodiments 145 to 148, wherein the influenza A virus is an influenza A virus of an H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or H18.

150. The method or the immunogenic composition for the use of embodiment 149, wherein the influenza A virus is an H5 HA subtype.

151. The method or the immunogenic composition for the use of embodiment 150, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues FIP and KIQLSTKNVINAEH APGGP YRL (SEQ ID NO:

109) ; b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PYQGKSS (SEQ ID NO: 124); c. the acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B

virus B/Yamagata/16/88 are substituted with amino acid residues KKNSTY (SEQ ID NO: 111); and/or d. the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NDAAMQT (SEQ ID NO: 113). 152. The method or the immunogenic composition for the use of embodiment 150 or 151, wherein the H5 subtype is influenza A/Vietnam/1203/04 (HALo) virus.

153. The method or the immunogenic composition for the use of embodiment 149, wherein the influenza A virus is an H8 HA subtype.

154. The method or the immunogenic composition for the use of embodiment 153, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues HIP and RIRLSTYNVINAET APGGP YRL (SEQ ID NO: 125); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

155. The method or the immunogenic composition for the use of 153 or 154, wherein the H8 subtype is influenza A/Mallard/Sweden/24/2002 virus.

156. The method or the immunogenic composition for the use of embodiment 149, wherein the influenza A virus is an HI 1 HA subtype.

157. The method or the immunogenic composition for the use of embodiment 156, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VINAERAPGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LIP and KIEL STSNVINAEVAPGGP YRL (SEQ ID NO: 129); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

158. The method or the immunogenic composition for the use of embodiment 156 or 157, wherein the HI 1 subtype is influenza A/northern shovel er/Netherlands/18/99 virus.

159. The method or the immunogenic composition for the use of embodiment 149, wherein the influenza A virus is an H12 HA subtype.

160. The method or the immunogenic composition for the use of embodiment 159, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VINAERAPGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues YIP and RIKL S TFN VINAET APGGP YRL (SEQ ID NO: 136); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues PTSDMQI (SEQ ID NO: 139).

161. The method or the immunogenic composition for the use of embodiment 159 or 160, wherein the H12 subtype is influenza A/mallard/interior Alaska/7MP0167/2007 virus.

162. The method or the immunogenic composition for the use of embodiment 149, wherein the influenza A virus is an HI 3 HA subtype.

163. The method or the immunogenic composition for the use of embodiment 162, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues VSTNMAK (SEQ ID NO: 135). 164. The method or the immunogenic composition for the use of embodiment 162 or 163, wherein the H13 subtype is influenza A/black headed gull/Sweden/1/99 virus.

165. The method or the immunogenic composition for the use of any one of

embodiments 145 to 164, wherein the HA globular head domain comprises one, two, or more amino acid substitutions outside of one, two, three, or all of the following: the 120 loop, the 150 loop, the 160 loop, and the 190 helix.

166. The method or the immunogenic composition for use of embodiment 136, 138, 139, 141, 142, or 144, wherein the HA stem domain polypeptide comprises an HA2 stem domain.

167. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 166, or the immunogenic composition for the use of any one of embodiments 92 to 93 or 100 to 114, or 118 to 166, wherein the liposomal adjuvant comprises a TLR4 agonist and saponin.

168. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 167, or the immunogenic composition for the use of any one of embodiments 92 to 93 or 100 to 114, or 118 to 167, wherein the TLR 4 agonist is a lipopolysaccharide.

169. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 167, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 167, wherein the TLR 4 agonist is a non-toxic derivative of lipid A.

170. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 167, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 167, wherein the TLR 4 agonist is a monophosphoryl lipid A.

171. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 167, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 167, wherein the TLR 4 agonist is 3-de-O-acylated monophosphoryl lipid A. 172. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 171, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 171, wherein the saponin is obtainable from Quillaja saponaria.

173. The method of any one of embodiments 79 to 104, 100 to 117, or 121 to 171, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 171, wherein the saponin comprises one or more of QS7, QS17, QS18 and QS21.

174. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 171, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 171, wherein the saponin comprises QS21.

175. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 171, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 171, wherein the TLR4 agonist is 3-O-desacyl-monophosphoiyl lipid A and the saponin is QS21.

176. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 175, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 175, wherein the liposomes are 30-200nm in size.

177. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 175, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 175, wherein the liposomes are 95-120 nm in size.

178. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 177, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 177, wherein the liposomes comprise l,2-dioleoyl-sn-glycero-3 -phosphatidylcholine (DOPC). 179. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 178, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 178, wherein the composition comprises a human dose volume of 0.05 ml to 1 ml of the lipososomal adjuvant.

180. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 178, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 178, wherein the composition comprises a human dose volume of 0.4 ml to 0.6 ml of the lipososomal adjuvant.

181. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 180, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 180, wherein the lipososomal adjuvant comprises 1-100 μg of the TLR4 agonist per human dose.

182. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 180, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 180, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose.

183. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 180, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 180, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist per human dose.

184. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 180, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 180, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose. 185. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 184, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 184, wherein the lipososomal adjuvant comprises 1-100 μg of the saponin per human dose.

186. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 180, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 180, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist and 20-30 μg of saponin per human dose.

187. The method of any one of embodiments 79 to 84, 100 to 117, or 121 to 180, or the immunogenic composition for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 180, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist and 40-60 μg of saponin per human dose.

188. The method of any one of embodiments 74, 115 to 117 or 121 to 187, or the immunogenic composition for the use of any one of embodiments 87 or 118 to 187, wherein the immunogenic composition is administered to the subject intramuscularly or subcutaneously.

189. The method of any one of embodiments 74, 115 to 117 or 121 to 187, or the immunogenic composition for the use of any one of embodiments 87 or 118 to 187, wherein the immunogenic composition is administered to the subject intramuscularly.

190. The method of any one of embodiments 75 to 86, 100 to 114 or 165 to 187, or the immunogenic compositions for the use of any one of embodiments 92 to 97 or 100 to 114 or 118 to 187, wherein the immunogenic compositions are administered to the subject intramuscularly or subcutaneously.

191. The method of any one of embodiments 75 to 86, 100 to 114 or 165 to 187, or the immunogenic compositions for the use of any one of embodiments 92 to 97 or 100 to 114, or 118 to 187, wherein the immunogenic compositions are administered to the subject intramuscularly. 192. The method of any one of embodiments 74 to 86, 100 to 117, 121 to 191, or the immunogenic composition for the use of any one of embodiments 87 to 97, 100 to 114, or 118 to 191, wherein the subject is a human adult.

193. The method of any one of embodiments 74 to 86, 100 to 117, 121 to 191, or the immunogenic composition for the use of any one of embodiments 87 to 97, 100 to 114, or 118 to 191, wherein the subject is an elderly human.

194. The method of any one of embodiments 74 to 86, 100 to 117, 121 to 191, or the immunogenic composition for the use of any one of embodiments 87 to 97, 100 to 114, or 118 to 191, wherein the subject is a human infant or human toddler.

195. A kit for immunizing against an influenza virus in a subject, comprising:

(a) a first container comprising an immunogenic composition, wherein the immunogenic composition comprises a chimeric influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and

(b) a second container comprising a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

196. A kit for immunizing against an influenza virus in a subject, comprising:

(a) a first container comprising an immunogenic composition, wherein the immunogenic composition comprises an inactivated influenza virus, wherein the inactivated influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and

(b) a second container comprising a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both. 197. A kit of immunizing against influenza vims in a subject (e.g., a human subject), comprising:

(a) a first container comprising an immunogenic composition, wherein the immunogenic composition comprises a split influenza virus and a liposomal adjuvant, wherein the split influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and

198. The kit of embodiment 195, wherein the immunogenic composition is a subunit vaccine.

199. The kit of any one of embodiments 195 to 199, wherein the influenza virus HA globular head domain is from one influenza A virus subtype and the HA stem domain polypeptide is from a different influenza A virus subtype.

200. The kit of embodiment 199, wherein the HA stem domain is from influenza A virus subtype HI .

201. The kit of embodiment 199, wherein the HA stem domain is from influenza A virus subtype H3.

202. The kit of any one of embodiments 199 to 201, wherein the influenza virus HA globular head domain is from influenza A virus H8.

203. The kit of any one of embodiments 199 to 201, wherein the influenza virus HA globular head domain is from influenza A virus H5. 204. The kit of any one of embodiments 199 to 201, wherein the influenza virus HA globular head domain is from influenza A virus subtype H4, H6, H7, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18.

205. The kit of any one of embodiments 195 to 198, wherein the influenza virus HA globular head domain is from one influenza virus species and the HA stem domain is from a different influenza virus species.

206. The kit of any one of embodiments 195 to 198, or the immunogenic composition for the use of any one of embodiments 118 to 121, wherein the influenza virus HA globular head domain is from an influenza A virus and the HA stem domain polypeptide is from an influenza B virus lineage.

207. The kit of embodiment 195 or 206 which further comprises an influenza virus neuraminidase (NA) polypeptide.

208. The kit of any one of embodiments 195 to 206, wherein 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 HAlN-term through Ap of an influenza A virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HA0 protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

209. The kit of any one of embodiments 195 to 205, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

210. The kit of any one of embodiments 195 to 205, wherein 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 HAlN-term through Ap of an influenza A virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, and wherein the influenza A virus HA globular head domain consists of the amino acid residues intervening Ap and Aq.

211. The kit of any one of embodiments 195 to 198 or 206, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C- terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering.

212. The kit of any one of embodiments 194 to 197 or 205, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

213. The kit of any one of embodiments 194 to 197 or 205, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C- terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

214. The kit of any one of embodiments 195 to 198 or 206, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C- terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N- terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq in the HAl C-terminal stem segment is substituted with a Cys.

215. The kit of any one of embodiments 195 to 198 or 206, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

216. The kit of any one of embodiments 195 to 198 or 206, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C- terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N- terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to

B/Yamagata/16/88 numbering, wherein Dq in the HAl C-terminal stem segment is substituted with a Cys, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

217. The kit of embodiment 208, 210, 211, 213, 214 or 216, wherein the HA stem domain polypeptide comprise an HA2 stem domain. 218. The kit of any one of embodiments 195 to 198, wherein the HA stem domain polypeptide is from an influenza B virus HA, and wherein the HA globular head domain is the globular head domain derived from the influenza B virus HA and comprises one, two, three or all of the following:

219. The kit of embodiment 218, wherein the chimeric influenza virus HA polypeptide further comprises the transmembrane domain and cytoplasmic tail domain of the influenza B virus HA. 220. The kit of embodiment 218 or 219, wherein the influenza B virus is of the

Yamagata lineage or of the Victoria lineage.

221. The kit of embodiment 218 or 219, wherein the influenza B virus is influenza B/Yamagata/16/88.

222. The kit of any one of embodiments 218 to 221, wherein the influenza A virus is an influenza A virus of an H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18.

223. The kit of embodiment 222, wherein the influenza A virus is an H5 HA subtype.

224. The kit of embodiment 223, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

109) ; b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NDAAMQT (SEQ ID NO: 113). 225. The kit of embodiment 223 or 224, wherein the H5 subtype is influenza A/Vietnam/1203/04 (HALo) virus.

226. The kit of embodiment 222, wherein the influenza A virus is an H8 HA subtype.

227. The kit of embodiment 226, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

228. The kit of embodiment 226 or 227, wherein the H8 subtype is influenza

A/Mallard/Sweden/24/2002 virus.

229. The kit of embodiment 222, wherein the influenza A virus is an HI 1 HA subtype.

230. The kit of embodiment 229, wherein the HA globular head domain compri two, three or all of the following: a. the amino acid residues TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LIP and KIEL S T SN VINAE V APGGP YRL (SEQ ID NO: 129); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

231. The kit of embodiment 229 or 230, wherein the HI 1 subtype is influenza

A/northern shovel er/Netherlands/18/99 virus.

232. The kit of embodiment 222, wherein the influenza A virus is an H12 HA subtype.

233. The kit of embodiment 232, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRLSTHNVINAERAPGGPYRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues YIP and RIKLSTFNVINAET APGGP YRL (SEQ ID NO: 136); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

234. The kit of embodiment 232 or 233, wherein the H12 subtype is influenza

A/mallard/interior Alaska/7MP0167/2007 virus.

235. The method or the immunogenic composition for the use of embodiment 222, wherein the influenza A virus is an HI 3 HA subtype.

236. The kit of embodiment 235, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

influenza B virus B/Yamagata/16/88 are substituted with amino acid residues VSTNMAK (SEQ ID NO: 135). 237. The kit of embodiment 235 or 236, wherein the H13 subtype is influenza A/black headed gull/Sweden/1/99 virus.

238. The kit of any one of embodiments 195 to 237, wherein the HA globular head domain comprises one, two, or more amino acid substitutions outside of one, two, three, or all of the following: the 120 loop, the 150 loop, the 160 loop, and the 190 helix.

239. The kit of any one of embodiments 195 to 238, wherein the liposomal adjuvant comprises a TLR4 agonist and saponin.

240. The kit of any one of embodiments 195 to 239, wherein the TLR 4 agonist is a lipopolysaccharide.

241. The kit of any one of embodiments 195 to 239, wherein the TLR 4 agonist is a non-toxic derivative of lipid A.

242. The kit of any one of embodiments 195 to 239, wherein the TLR 4 agonist is a monophosphoryl lipid A.

243. The kit of any one of embodiments 195 to 239, wherein the TLR 4 agonist is 3- de-O-acylated monophosphoryl lipid A.

244. The kit of any one of embodiments 195 to 243, wherein the saponin is obtainable from Quillaja saponaria.

245. The kit of any one of embodiments 195 to 243, wherein the saponin comprises one or more of QS7, QS17, QS18 and QS21.

246. The kit of any one of embodiments 195 to 243, wherein the saponin comprises QS21. 247. The kit of any one of embodiments 195 to 239, wherein the TLR4 agonist is 3-0- desacyl-monophosphoryl lipid A and the saponin is QS21.

248. The method kit of any one of embodiments 195 to 247, wherein the liposomes are 30-200nm in size.

249. The kit of any one of embodiments 195 to 247, wherein the liposomes are 95-120 nm in size.

250. The kit of any one of embodiments 195 to 249, wherein the liposomes comprise l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC).

251. The kit of any one of embodiments 195 to 250, wherein the composition comprises a human dose volume of 0.05 ml to 1 ml of the lipososomal adjuvant.

252. The kit of any one of embodiments 195 to 250, wherein the composition comprises a human dose volume of 0.4 ml to 0.6 ml of the lipososomal adjuvant.

253. The kit of any one of embodiments 195 to 252, wherein the lipososomal adjuvant comprises 1-100 μ of the TLR4 agonist per human dose.

254. The kit of any one of embodiments 195 to 252, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose.

255. The kit of any one of embodiments 195 to 252, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist per human dose.

256. The kit of any one of embodiments 195 to 252, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose. 257. The kit of any one of embodiments 195 to 256, wherein the lipososomal adjuvant comprises 1-100 μ of the saponin per human dose.

258. The kit of any one of embodiments 195 to 252, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist and 20-30 μg of saponin per human dose.

259. The kit of any one of embodiments 195 to 252, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist and 40-60 μg of saponin per human dose.

260. A liposomal adjuvant for use with a chimeric influenza virus hemagglutinin (HA) polypeptide for immunizing against influenza virus, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

261. A chimeric influenza virus hemagglutinin (HA) polypeptide for use with a liposomal adjuvant in the immunization of a subject against influenza virus, wherein the chimeric influenza virus HA polypeptide 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 polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

8. EQUIVALENTS

[00688] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[00689] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00690] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED:

4. A method for immunizing against an influenza virus in a subject, comprising:

influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

5. A method for immunizing against an influenza virus in a subject, comprising:

6. A method of immunizing against influenza virus in a human subject, comprising:

7. An immunogenic composition for use in a method for immunizing against an influenza virus in a subject, the method comprising:

(a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises a chimeric influenza virus hemagglutinin (HA) polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and (b) administering to the subject a liposomal adjuvant, wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

8. An immunogenic composition for use in a method for immunizing against an influenza virus in a subject, the method comprising:

9. An immunogenic composition for use in a method of immunizing against influenza virus in a human subject, the method comprising:

(a) administering to the subject the immunogenic composition, wherein the immunogenic composition comprises a split influenza virus and a liposomal adjuvant, wherein the split influenza virus comprises a chimeric influenza virus HA polypeptide, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide; and

10. A kit for immunizing against an influenza virus in a subject, comprising:

11. A kit for immunizing against an influenza virus in a subject, comprising:

12. A kit of immunizing against influenza virus in a human subject, comprising:

13. An influenza virus chimeric hemagglutinin (HA) polypeptide for use with a liposomal adjuvant for immunizing a subject against influenza virus, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

14. A liposomal adjuvant for use with an influenza virus chimeric hemagglutinin (HA) polypeptide for immunizing a subject against influenza virus, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain polypeptide, and wherein the influenza virus HA globular head domain is heterologous to the HA stem domain polypeptide, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both.

15. The immunogenic composition of claim 1, the method of claim 4, the immunogenic composition for the use of claim 7, the kit of claim 10, the chimeric HA polypeptide of claim 13, or the liposomal adjuvant of claim 14, which is a subunit vaccine.

16. The immunogenic composition of any one of claims 1 to 3 or 15, the method of any one of claims 4 to 6 or 15, the immunogenic composition for the use of any one of claims 7 to 9 or 15, the kit of any one of claims 10 to 12 or 15, the chimeric HA polypeptide of claim 13 or 15, or the liposomal adjuvant of claim 14 or 15, wherein the influenza virus HA globular head domain is from one influenza A virus subtype and the HA stem domain polypeptide is from a different influenza A virus subtype.

17. The immunogenic composition of claim 16, the method of claim 16, the immunogenic composition for the use of claim 16, the kit of claim 16, the chimeric HA polypeptide of claim 16, or the liposomal adjuvant of claim 16, wherein the HA stem domain is from influenza A virus subtype HI .

18. The immunogenic composition of claim 16, the method of claim 16, the immunogenic composition for the use of claim 16, the kit of claim 16, the chimeric HA polypeptide of claim 16, or the liposomal adjuvant of claim 16, wherein the HA stem domain is from influenza A virus subtype H3.

19. The immunogenic composition of any one of claims 16 to 18, the method of any one of claims 16 to 18, the immunogenic composition for the use of any one of claims 16 to 18, the kit of any one of claims 16 to 18, the chimeric HA polypeptide of any one of claims 16 to 18, or the liposomal adjuvant of any one of claims 16 to 18, wherein the influenza virus HA globular head domain is from influenza A virus subtype H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18.

20. The immunogenic composition of any one of claims 1 to 3 or 15, the method of any one of claims 4 to 6 or 15, the immunogenic composition for the use of any one of claims 7 to 9 or 15, the kit of any one of claims 10 to 12 or 15, the chimeric HA polypeptide of claim 13 or 15, or the liposomal adjuvant of claim 14 or 15, wherein the influenza virus HA globular head domain is from one influenza virus species and the HA stem domain is from a different influenza virus species.

21. The immunogenic composition of any one of claims 1 to 3 or 15, the method of any one of claims 4 to 6 or 15, the immunogenic composition for the use of any one of claims 7 to 9 or 15, the kit of any one of claims 10 to 12 or 15, the chimeric HA polypeptide of claim 13 or 15, or the liposomal adjuvant of claim 14 or 15, wherein the influenza virus HA globular head domain is from one influenza B virus lineage and the HA stem domain polypeptide is from a different influenza B virus lineage.

22. The immunogenic composition of any one of claims 1 to 3 or 15, the method of any one of claims 4 to 6 or 15, the immunogenic composition for the use of any one of claims 7 to 9 or 15, the kit of any one of claims 10 to 12 or 15, the chimeric HA polypeptide of claim 13 or 15, or the liposomal adjuvant of claim 14 or 15, wherein the influenza virus HA globular head domain is from an influenza A virus and the HA stem domain polypeptide is from one influenza B virus lineage.

23. The immunogenic composition of any one of claims 1 to 3 or 15 to 20, the method of any one of claims 4 to 6 or 15 to 20, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 20, the kit of any one of claims 10 to 12 or 15 to 20, the chimeric HA polypeptide of any one of claims 13 or 15 to 20, or the liposomal adjuvant of any one of claims 14 to 20, wherein the HA stem domain polypeptide 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 HAlN-term through Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HA1 domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HA1 domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

24. The immunogenic composition of any one of claims 1 to 3 or 15 to 20, the method of any one of claims 4 to 6 or 15 to 20, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 20, the kit of any one of claims 10 to 12 or 15 to 20, the chimeric HA polypeptide of any one of claims 13 or 15 to 20, or the liposomal adjuvant of any one of claims 14 to 20, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

25. The immunogenic composition of any one of claims 1 to 3 or 15 to 20, the method of any one of claims 4 to 6 or 15 to 20, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 20, the kit of any one of claims 10 to 12 or 15 to 20, the chimeric HA polypeptide of any one of claims 13 or 15 to 20, or the liposomal adjuvant of any one of claims 14 to 20, wherein the HA stem domain polypeptide 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 HAlN-term through Ap of an influenza A virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Aq through HAlc-term of an influenza A virus hemagglutinin HA1 domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HA1 domain, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq.

26. The immunogenic composition of any one of claims 1 to 3, 15, 21 or 22, the method of any one of claims 4 to 6, 15, 21 or 22, the immunogenic composition for the use of any one of claims 7 to 9, 15, 21 or 22, the kit of any one of claims 10 to 12, 15 or 22, the chimeric HA polypeptide of claim 13, 15, 21 or 22, or the liposomal adjuvant of claim 14, 15, 21 or 22, wherein the HA stem domain polypeptide 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 HAlN-term through Dp of an influenza B virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HA1 domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HA1 domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HA1 domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HA1 domain according to B/Yamagata/16/88 numbering.

27. The immunogenic composition of any one of claims 1 to 3, 15 or 22, the method of any one of claims 4 to 6, 15 or 22, the immunogenic composition for the use of any one of claims 7 to 9, 15 or 22, the kit of any one of claims 10 to 12, 15 or 22, the chimeric HA polypeptide of claim 13, 15 or 22, or the liposomal adjuvant of claim 14, 15 or 22, wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

28. The immunogenic composition of any one of claims 1 to 3, 15 or 22, the method of any one of claims 4 to 6, 15 or 22, the immunogenic composition for the use of any one of claims 7 to 9, 15 or 22, the kit of any one of claims 10 to 12, 15 or 22, the chimeric HA polypeptide of claim 13, 15 or 22, or the liposomal adjuvant of claim 14, 15 or 22, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HAl domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HAl domain according to H3 numbering.

29. The immunogenic composition of any one of claims 1 to 3, 15 or 22, the method of any one of claims 4 to 6, 15 or 22, the immunogenic composition for the use of any one of claims 7 to 9, 15 or 22, the kit of any one of claims 10 to 12, 15 or 22, the chimeric HA polypeptide of claim 13, 15 or 22, or the liposomal adjuvant of claim 14, 15 or 22, wherein 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 HAlN-term through Dp of an influenza B virus hemagglutinin HAl domain, and wherein the HAl C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HAl domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HAl domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, wherein Dp in the N- terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HAl domain according to B/Yamagata/16/88 numbering, and wherein Dq in the HA1 C-terminal stem segment is substituted with a Cys.

30. The immunogenic composition of any one of claims 1 to 3, 15 or 22, the method of any one of claims 4 to 6, 15 or 22, the immunogenic composition for the use of any one of claims 7 to 9, 15 or 22, the kit of any one of claims 10 to 12, 15 or 22, the chimeric HA polypeptide of claim 13, 15 or 22, or the liposomal adjuvant of claim 14, 15 or 22, wherein the HA stem domain polypeptide 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 HAlN-term through Dp of an influenza B virus hemagglutinin HA1 domain, and wherein the HA1 C-terminal stem segment consists of amino acid residues Dq through HAlc-term of an influenza B virus hemagglutinin HA1 domain, wherein HAlN-term is the N-terminal amino acid of a mature HAO protein lacking a signal peptide, wherein HAlc-term is the C-terminal amino acid of the HA1 domain, wherein Dp is Ala that corresponds to amino acid position 57 of an influenza B virus hemagglutinin HA1 domain according to B/Yamagata/16/88 numbering, wherein Dp in the N- terminal stem segment is substituted with a Cys, wherein Dq is Ala that corresponds to amino acid position 306 of an influenza B virus hemagglutinin HA1 domain according to

B/Yamagata/16/88 numbering, wherein Dq in the HA1 C-terminal stem segment is substituted with a Cys, and wherein the influenza virus HA globular head domain consists of the amino acid residues intervening Ap and Aq, wherein Ap is Cys that corresponds to amino acid position 52 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering, wherein Aq is Cys that corresponds to amino acid position 277 of an influenza A virus hemagglutinin HA1 domain according to H3 numbering.

31. The immunogenic composition of any one of claims 1 to 3 or 15 to 30, the method of any one of claims 4 to 6 or 15 to 30, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 30, the kit of any one of claims 10 to 12 or 15 to 30, the chimeric HA polypeptide of any one of claims 13 or 15 to 30, or the liposomal adjuvant of any one of claims 14 or 15 to 30, wherein the influenza virus HA stem domain further comprises the stem domain of an influenza virus HA2 domain.

32. The immunogenic composition of any one of claims 1 to 3 or 15 to 30, the method of any one of claims 4 to 6 or 15 to 30, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 30, the kit of any one of claims 10 to 12 or 15 to 30, the chimeric HA polypeptide of any one of claims 13 or 15 to 30, or the liposomal adjuvant of any one of claims 14 or 15 to 30, wherein the influenza virus HA stem domain further comprises an influenza virus HA2 domain lacking the cytoplasmic domain and transmembrane domain.

33. The immunogenic composition of any one of claims 1 to 3 or 15 to 32, the method of any one of claims 4 to 6 or 15 to 32, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 32, the kit of any one of claims 10 to 12 or 15 to 32, the chimeric HA polypeptide of any one of claims 13 or 15 to 32, or the liposomal adjuvant of any one of claims 14 or 15 to 32, wherein the HA2 domain is from the same strain as the HA1 N-terminal stem segment and HA1 C-terminal segment.

34. The immunogenic composition of any one of claims 1 to 3 or 15, the method of any one of claims 4 to 6 or 15, the immunogenic composition for the use of any one of claims 7 to 9 or 15, the kit of any one of claims 10 to 12 or 15, the chimeric HA polypeptide of claim 13 or 15, or the liposomal adjuvant of claim 14 or 15, wherein the HA stem domain polypeptide is from an influenza B virus HA, and wherein the HA globular head domain is the globular head domain derived from the influenza B virus HA and comprises one, two, three or all of the following: a. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions within the 120 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in the 120 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA; b. 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid substitutions within the 150 loop of the globular head domain of the influenza B virus HA, wherein the amino acid substitutions substitute 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid residues in the 150 loop of the globular head of the influenza B virus HA with amino acid residues found in a corresponding region of the globular domain of an influenza A virus HA;

35. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 34, wherein the chimeric influenza virus HA polypeptide further comprises the transmembrane domain and cytoplasmic tail domain of the influenza B virus HA.

36. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 34 or 35, wherein the influenza B virus is of the Yamagata lineage or of the Victoria lineage.

37. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 34 or 35, wherein the influenza B virus is influenza B/Yamagata/16/88.

38. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of any one of claims 34 to 37, wherein the influenza A virus is an influenza A virus of an H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16, H17, or H18.

39. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 38, wherein the influenza A virus is an H5 HA subtype.

40. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 39, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

109) ; b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

41. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 39 or 40, wherein the H5 subtype is influenza A/Vietnam/1203/04 (HALo) virus.

42. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 38, wherein the influenza A virus is an H8 HA subtype.

43. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 42, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

44. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 42 or 43, wherein the H8 subtype is influenza A/Mallard/Sweden/24/2002 virus.

45. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 38, wherein the influenza A virus is an HI 1 HA subtype.

46. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 45, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VINAERAPGGP YRL (SEQ ID NO:

47. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 45 or 46, wherein the HI 1 subtype is influenza A/northern shovel er/Netherlands/18/99 virus.

48. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 38, wherein the influenza A virus is an H12 HA subtype.

49. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 48, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VINAERAPGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues YIP and RIKL S TFN VIN AET APGGP YRL (SEQ ID NO: 136); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NNTSNQGS (SEQ ID NO: 137); c. the amino acid residues RDNKTA (SEQ ID NO: 110) in the 160 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues LKSGQF (SEQ ID NO: 138); and/or d. the amino acid residues NKNQMKN (SEQ ID NO: 112) in the 190 helix of

50. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 48 or 49, wherein the H12 subtype is influenza A/mallard/interior Alaska/7MP0167/2007 virus.

51. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 38, wherein the influenza A virus is an HI 3 HA subtype.

52. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 51, wherein the HA globular head domain comprises one, two, three or all of the following: a. the amino acid residues TIP and NIRL S THN VIN AER APGGP YRL (SEQ ID NO:

108) in the 120 loop of influenza B virus B/Yamagata/16/88 are substituted with amino acid residues NIP and RIELSTHNVINAEV APGGP YRL (SEQ ID NO: 201); b. the amino acid residues PNVTSRNG (SEQ ID NO: 123) in the 150 loop of

53. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of claim 51 or 52, wherein the H13 subtype is influenza A/black headed gull/Sweden/1/99 virus.

54. The immunogenic composition, the method, the immunogenic composition for the use, the kit, the chimeric HA polypeptide, or the liposomal adjuvant of any one of claims 34 to 53, wherein the HA globular head domain comprises one, two, or more amino acid substitutions outside of one, two, three, or all of the following: the 120 loop, the 150 loop, the 160 loop, and the 190 helix.

55. The immunogenic composition of any one of claims 1 to 3 or 15 to 54, the method of any one of claims 4 to 6 or 15 to 54, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 54, the kit of any one of claims 10 to 12 or 15 to 54, the chimeric HA polypeptide of any one of claims 13 or 15 to 54, or the liposomal adjuvant of any one of claims 14 or 15 to 54, wherein the liposomal adjuvant comprises a TLR4 agonist and saponin.

56. The immunogenic composition of any one of claims 1 to 3 or 15 to 55, the method of any one of claims 4 to 6 or 15 to 55, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 55, the kit of any one of claims 10 to 12 or 15 to 55, the chimeric HA polypeptide of any one of claims 13 or 15 to 55, or the liposomal adjuvant of any one of claims 14 or 15 to 55, wherein the TLR 4 agonist is a lipopolysaccharide.

57. The immunogenic composition of any one of claims 1 to 3 or 15 to 55, the method of any one of claims 4 to 6 or 15 to 55, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 55, the kit of any one of claims 10 to 12 or 15 to 55, the chimeric HA polypeptide of any one of claims 13 or 15 to 55, or the liposomal adjuvant of any one of claims 14 or 15 to 55, wherein the TLR 4 agonist is a non-toxic derivative of lipid A.

58. The immunogenic composition of any one of claims 1 to 3 or 15 to 55, the method of any one of claims 4 to 6 or 15 to 55, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 55, the kit of any one of claims 10 to 12 or 15 to 55, the chimeric HA polypeptide of any one of claims 13 or 15 to 55, or the liposomal adjuvant of any one of claims 14 or 15 to 55, wherein the TLR 4 agonist is a monophosphoryl lipid A.

59. The immunogenic composition of any one of claims 1 to 3 or 15 to 55, the method of any one of claims 4 to 6 or 15 to 55, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 55, the kit of any one of claims 10 to 12 or 15 to 55, the chimeric HA polypeptide of any one of claims 13 or 15 to 55, or the liposomal adjuvant of any one of claims 14 or 15 to 55, wherein the TLR 4 agonist is 3-de-O-acylated monophosphoryl lipid A.

60. The immunogenic composition of any one of claims 1 to 3 or 15 to 59, the method of any one of claims 4 to 6 or 15 to 59, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 59, the kit of any one of claims 10 to 12 or 15 to 59, the chimeric HA polypeptide of any one of claims 13 or 15 to 59, or the liposomal adjuvant of any one of claims 14 or 15 to 59, wherein the saponin is obtainable from Quillaja saponaria.

61. The immunogenic composition of any one of claims 1 to 3 or 15 to 59, the method of any one of claims 4 to 6 or 15 to 59, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 59, the kit of any one of claims 10 to 12 or 15 to 59, the chimeric HA polypeptide of any one of claims 13 or 15 to 59, or the liposomal adjuvant of any one of claims 14 or 15 to 59, wherein the saponin comprises one or more of QS7, QS17, QS18 and QS21.

62. The immunogenic composition of any one of claims 1 to 3 or 15 to 59, the method of any one of claims 4 to 6 or 15 to 59, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 59, the kit of any one of claims 10 to 12 or 15 to 59, the chimeric HA polypeptide of any one of claims 13 or 15 to 59, or the liposomal adjuvant of any one of claims 14 or 15 to 59, wherein the saponin comprises QS21.

63. The immunogenic composition of any one of claims 1 to 3 or 15 to 55, the method of any one of claims 4 to 6 or 15 to 55, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 55, the kit of any one of claims 10 to 12 or 15 to 55, the chimeric HA polypeptide of any one of claims 13 or 15 to 55, or the liposomal adjuvant of any one of claims 14 or 15 to 55, wherein the TLR4 agonist is 3-O-desacyl-monophosphoiyl lipid A and the saponin is QS21.

64. The immunogenic composition of any one of claims 1 to 3 or 15 to 63, the method of any one of claims 4 to 6 or 15 to 63, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 63, the kit of any one of claims 10 to 12 or 15 to 63, the chimeric HA polypeptide of any one of claims 13 or 15 to 63, or the liposomal adjuvant of any one of claims 14 or 15 to 63, wherein the liposomes are 30-200 nm in size.

65. The immunogenic composition of any one of claims 1 to 3 or 15 to 63, the method of any one of claims 4 to 6 or 15 to 63, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 63, or the kit of any one of claims 10 to 12 or 15 to 63, the chimeric HA polypeptide of any one of claims 13 or 15 to 63, or the liposomal adjuvant of any one of claims 14 or 15 to 63, wherein the liposomes are 95-120 nm in size.

66. The immunogenic composition of any one of claims 1 to 3 or 15 to 65, the method of any one of claims 4 to 6 or 15 to 65, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 65, or the kit of any one of claims 10 to 12 or 15 to 65, the chimeric HA polypeptide of any one of claims 13 or 15 to 65, or the liposomal adjuvant of any one of claims 14 or 15 to 65, wherein the liposomes comprise l,2-dioleoyl-sn-glycero-3 -phosphatidyl choline (DOPC).

67. The immunogenic composition of any one of claims 1 to 3 or 15 to 66, the method of any one of claims 4 to 6 or 15 to 66, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 66, or the kit of any one of claims 10 to 12 or 15 to 66, wherein the composition comprises a human dose volume of 0.05 ml to 1 ml of the lipososomal adjuvant.

68. The immunogenic composition of any one of claims 1 to 3 or 15 to 66, the method of any one of claims 4 to 6 or 15 to 66, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 66, or the kit of any one of claims 10 to 12 or 15 to 66, wherein the composition comprises a human dose volume of 0.4 ml to 0.6 ml of the lipososomal adjuvant.

69. The immunogenic composition of any one of claims 1 to 3 or 15 to 68, the method of any one of claims 4 to 6 or 15 to 68, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 68, or the kit of any one of claims 10 to 12 or 15 to 68, wherein the lipososomal adjuvant comprises 1-100 μg of the TLR4 agonist per human dose.

70. The immunogenic composition of any one of claims 1 to 3 or 15 to 68, the method of any one of claims 4 to 6 or 15 to 68, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 68, or the kit of any one of claims 10 to 12 or 15 to 68, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose.

71. The immunogenic composition of any one of claims 1 to 3 or 15 to 68, the method of any one of claims 4 to 6 or 15 to 68, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 68, or the kit of any one of claims 10 to 12 or 15 to 68, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist per human dose.

72. The immunogenic composition of any one of claims 1 to 3 or 15 to 68, the method of any one of claims 4 to 6 or 15 to 68, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 68, or the kit of any one of claims 10 to 12 or 15 to 68, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist per human dose.

73. The immunogenic composition of any one of claims 1 to 3 or 15 to 72, the method of any one of claims 4 to 6 or 15 to 72, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 72, or the kit of any one of claims 10 to 12 or 15 to 72, wherein the lipososomal adjuvant comprises 1-100 μg of the saponin per human dose.

74. The The immunogenic composition of any one of claims 1 to 3 or 15 to 72, the method of any one of claims 4 to 6 or 15 to 72, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 72, or the kit of any one of claims 10 to 12 or 15 to 72, wherein the lipososomal adjuvant comprises 20-30 μg of the TLR4 agonist and 20-30 μg of saponin per human dose.

75. The immunogenic composition of any one of claims 1 to 3 or 15 to 70, the method of any one of claims 4 to 6 or 15 to 70, the immunogenic composition for the use of any one of claims 7 to 9 or 15 to 70, or the kit of any one of claims 10 to 12 or 15 to 70, wherein the lipososomal adjuvant comprises 40-60 μg of the TLR4 agonist and 40-60 μg of saponin per human dose.

76. A method for immunizing a subject against influenza virus, comprising administering to the subject an immunogenic composition of any one of claims 1 to 3 or 15 to 75.

77. The immunogenic composition of any one of claims 1 to 3 or 15 to 75 for use in immunizing a subject against influenza virus.

78. A method for immunizing against influenza virus in a human subject, comprising: (a) administering to the subject a first immunogenic composition comprising a first inactivated influenza A virus and a liposomal adjuvant, wherein the first inactivated influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and (b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second inactivated influenza A virus and the liposomal adjuvant, wherein the second inactivated influenza A virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype.

79. A method for immunizing against influenza virus in a human subject, comprising: (a) administering to the subject a first immunogenic composition comprising a first split influenza A virus and a liposomal adjuvant, wherein the first split influenza A virus comprises a first chimeric influenza virus HA polypeptide, wherein the first chimeric influenza virus HA polypeptide comprises a first influenza virus HA globular head domain and a first influenza virus HA stem domain polypeptide, wherein the first influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H5 subtype and the first HA stem domain polypeptide is the HA stem domain polypeptide of an influenza A virus HI subtype, and wherein the liposomal adjuvant comprises liposomes and either a Toll-like receptor 4 (TLR4) agonist, saponin or both; and (b) a certain time after the administration of the first immunogenic composition, administering to the subject a second immunogenic composition, wherein the second immunogenic composition comprises a second split influenza A virus and the liposomal adjuvant, wherein the second split influenza A virus comprises a second chimeric influenza virus HA polypeptide, wherein the second influenza virus HA polypeptide comprises a second influenza virus HA globular head domain and the first influenza virus HA stem domain polypeptide, and wherein the second influenza virus HA globular head domain is the HA globular head domain of an influenza A virus H8 subtype.

80. A method for immunizing a subject against influenza virus comprising: (A)

administering to the subject a first immunogenic composition comprising a first chimeric HA polypeptide, a second chimeric HA polypeptide and a third chimeric HA polypeptide, wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A vims of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1, and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2, wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of a first influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the first influenza B virus HA globular head domain, and the third HA stem domain comprises the HA stem domain of the first influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain; (B) administering to the subject a second immunogenic composition comprising a fourth chimeric HA polypeptide, a fifth chimeric HA polypeptide and a sixth chimeric HA polypeptide, wherein (i) the fourth chimeric HA polypeptide comprises a fourth HA globular head domain of a fifth influenza A virus of a fifth HA subtype and a fourth HA stem domain of the second influenza A virus of the second subtype belonging to group 1, and wherein the fourth HA globular head domain is heterologous to the fourth HA stem domain, (ii) the fifth chimeric HA polypeptide comprises a fifth HA globular head domain of a sixth influenza A virus of a sixth HA subtype and a fifth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2, wherein the fifth HA globular head domain is heterologous to the fifth HA stem domain, wherein the second, fourth, fifth and sixth HA subtypes are different from each other, and (iii) the sixth chimeric HA polypeptide comprises a sixth HA globular head domain and a sixth HA stem domain, wherein the sixth HA globular head domain comprises the HA globular head domain of a second influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the second influenza B virus HA globular head domain, and the sixth HA stem domain comprises the HA stem domain of the second influenza B virus, wherein the sixth HA globular head domain is heterologous to the sixth HA stem domain; and (C) administering to the subject a third immunogenic composition comprising a seventh chimeric HA polypeptide, an eighth chimeric HA polypeptide and a ninth chimeric HA polypeptide, wherein (i) the seventh chimeric HA polypeptide comprises a seventh HA globular head domain of a seventh influenza A virus of a seventh HA subtype and a seventh HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1, and wherein the seventh HA globular head domain is heterologous to the seventh HA stem domain, (ii) the eighth chimeric HA polypeptide comprises an eighth HA globular head domain of an eighth influenza A virus of an eighth HA subtype and an eighth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2, wherein the eighth HA globular head domain is heterologous to the eighth HA stem domain, wherein the second, fourth, seventh and eighth HA subtypes are different from each other, and (iii) the ninth chimeric HA polypeptide comprises a ninth HA globular head domain and a ninth HA stem domain, wherein the ninth HA globular head domain comprises the HA globular head domain of a third influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the third influenza B virus HA globular head domain, and the ninth HA stem domain comprises the HA stem domain of the third influenza B virus, wherein the ninth HA globular head domain is heterologous to the ninth HA stem domain.

81. A method for immunizing a subject against influenza virus comprising: (A) administering to the subject a first immunogenic composition comprising a first chimeric HA polypeptide, a second chimeric HA polypeptide, a third chimeric HA polypeptide and an adjuvant, wherein (i) the first chimeric HA polypeptide comprises a first HA globular head domain of a first influenza A virus of a first HA subtype and a first HA stem domain of a second influenza A virus of a second HA subtype belonging to group 1, and wherein the first HA globular head domain is heterologous to the first HA stem domain, (ii) the second chimeric HA polypeptide comprises a second HA globular head domain of a third influenza A virus of a third HA subtype and a second HA stem domain of a fourth influenza A virus of a fourth HA subtype belonging to group 2, wherein the second HA globular head domain is heterologous to the second HA stem domain, wherein the first, second, third, and fourth HA subtypes are different from each other, and (iii) the third chimeric HA polypeptide comprises a third HA globular head domain and a third HA stem domain, wherein the third HA globular head domain comprises the HA globular head domain of a first influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the first influenza B virus HA globular head domain, and the third HA stem domain comprise the HA stem domain of the first influenza B virus, wherein the third HA globular head domain is heterologous to the third HA stem domain; (B) administering to the subject a second immunogenic composition comprising a fourth chimeric HA polypeptide, a fifth chimeric HA polypeptide, a sixth chimeric HA polypeptide and an adjuvant, wherein (i) the fourth chimeric HA polypeptide comprises a fourth HA globular head domain of a fifth influenza A virus of a fifth HA subtype and a fourth HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1, and wherein the fourth HA globular head domain is heterologous to the fourth HA stem domain, (ii) the fifth chimeric HA polypeptide comprises a fifth HA globular head domain of a sixth influenza A virus of a sixth HA subtype and a fifth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2, wherein the fifth HA globular head domain is heterologous to the fith HA stem domain, wherein the second, fourth, fifth and sixth HA subtypes are different from each other, and (iii) the sixth chimeric HA polypeptide comprises a sixth HA globular head domain and a sixth HA stem domain, wherein the sixth HA globular head domain comprises the HA globular head domain of a second influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the second influenza B virus HA globular head domain, and the sixth HA stem domain comprises the HA stem domain of the second influenza B virus, wherein the sixth HA globular head domain is heterologous to the sixth HA stem domain; and (C) administering to the subject a third immunogenic composition comprising a seventh chimeric HA polypeptide, an eighth chimeric HA polypeptide, a ninth chimeric HA polypeptide and an adjuvant, wherein (i) the seventh chimeric HA polypeptide comprises a seventh HA globular head domain of a seventh influenza A virus of a seventh HA subtype and a seventh HA stem domain of the second influenza A virus of the second HA subtype belonging to group 1, and wherein the seventh HA globular head domain is heterologous to the seventh HA stem domain, (ii) the eighth chimeric HA polypeptide comprises an eighth HA globular head domain of an eighth influenza A virus of an eighth HA subtype and an eighth HA stem domain of the fourth influenza A virus of the fourth HA subtype belonging to group 2, wherein the eighth HA globular head domain is heterologous to the eighth HA stem domain, wherein the second, fourth, seventh and eighth HA subtypes are different from each other, and (iii) the ninth chimeric HA polypeptide comprises a ninth HA globular head domain and a ninth HA stem domain, wherein the ninth HA globular head domain comprises the HA globular head domain of a third influenza B virus having one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions in the one, two, three or all of the 120 loop, 150 loop, 160 loop and 190 helix of the third influenza B virus HA globular head domain, and the ninth HA stem domain comprising the HA stem domain of the third influenza B virus, wherein the ninth HA globular head domain is heterologous to the ninth HA stem domain.

82. A method of immunizing a subject against influenza virus comprising administering an immunogenic composition comprising a chimeric influenza virus HA polypeptide and a liposomal adjuvant, wherein the chimeric influenza virus HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain, wherein the globular head domain is heterologous to the stem domain, wherein the liposomal adjuvant comprises liposomes and either a TLR-4 agonist, saponin or both, and wherein the composition in an animal induces influenza virus-specific T cells in the lung.

83. An immunogenic composition comprising a chimeric influenza virus HA polypeptide and a liposomal adjuvant, for use in a method of immunizing a subject against influenza virus, wherein the method comprises administering the chimeric HA polypeptide to the subject, wherein chimeric HA polypeptide comprises an influenza virus HA globular head domain and an influenza virus HA stem domain, wherein the globular head domain is heterologous to the stem domain, wherein the liposomal adjuvant comprises liposomes and either a TLR-4 agonist, saponin or both, and wherein the composition in an animal induces influenza virus-specific T cells in the lung.

84. The method of any one of claims 4 to 6, 15 to 76, or 78 to 81, or the immunogenic composition for the use of any one of claims 7 to 9, 15 to 76, 82, or 83, wherein the

immunogenic composition is administered to the subject intramuscularly or subcutaneously.

85. The method of any one of claims 4 to 6, 15 to 76, or 78 to 81, or the immunogenic composition for the use of any one of claims 7 to 9, 15 to 75, 77, 82, or 83, wherein the immunogenic composition is administered to the subject intramuscularly.

86. The method of any one of claims 4 to 6, 15 to 76, 78 to 81, 84, or 85, or the immunogenic composition for the use of any one of claims 7 to 9, 15 to 75, 77, or 82 to 85, wherein the human subject is a human adult.

87. The method of any one of claims 4 to 6, 15 to 76, 78 to 81, 84, or 85, or the immunogenic composition for the use of any one of claims 7 to 9, 15 to 75, 77, or 82 to 85, wherein the human subject is an elderly human.

88. The method of any one of claims 4 to 6, 15 to 76, 78 to 81, 84, or 85, or the immunogenic composition for the use of any one of claims 7 to 9, 15 to 75, 77, or 82 to 85, wherein the human subject is a human infant or human toddler.

89. A kit comprising the immunogenic composition of any one of claims 1 to 3 or 15 to 75, in one or more containers.