Novel influenza hemagglutinin protein-based vaccines

Abstract

Novel vaccines are provided that elicit broadly neutralizing anti-influenza antibodies. Some vaccines comprise nanoparticles that display hemagglutinin trimers from influenza virus on their surface. The nanoparticles comprise fusion proteins comprising a monomeric subunit of ferritin joined to at least a portion of an influenza hemagglutinin protein. Some portions comprise the ectodomain while some portions are limited to the stem region. The fusion proteins self-assemble to form the hemagglutinin-displaying nanoparticles. Some vaccines comprise only the stem region of an influenza hemagglutinin protein joined to a trimerization domain. Such vaccines can be used to vaccinate an individual against infection by heterologous influenza viruses and influenza virus that are antigenically divergent from the virus from which the nanoparticle hemagglutinin protein was obtained. Also provided are fusion proteins and nucleic acid molecules encoding such proteins. Finally, also provided are assays using nanoparticles of the invention to detect anti-influenza antibodies.

What is claimed: 
     
         1 . A method for detecting anti-influenza virus antibodies in a sample, the method comprising:
 a. contacting at least a portion of the sample with a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to at least one epitope from an influenza hemagglutinin (HA) protein such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the at least a portion of the sample and the nanoparticle are contacted under conditions suitable for forming a complex between the nanoparticle and the anti-influenza virus antibodies, if present; and,   b. analyzing the contacted sample for the presence of a nanoparticle/antibody complex, wherein the presence of such a complex indicates the sample contains anti-influenza antibodies.   
     
     
         2 . The method of  claim 1 , wherein the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin and a mammalian ferritin. 
     
     
         3 . The nanoparticle of  claim 1 , wherein the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, where in the fusion protein is capable of self-assembling into nanoparticles. 
     
     
         4 . The nanoparticle of  claim 1 , wherein the hemagglutinin protein is from an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B). 
     
     
         5 . The nanoparticle of  claim 1 , wherein the fusion protein comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus. 
     
     
         6 . The nanoparticle of  claim 1 , wherein the nanoparticle comprises a second fusion protein comprising a second influenza hemagglutinin protein, wherein the first and second influenza hemagglutinin proteins are from different types, sub-types or strains of influenza viruses. 
     
     
         7 . The method of  claim 1 , wherein the step of analyzing comprises an assay selected from the group consisting of a hemagglutinin inhibition assay, an immunodiffusion assay, an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a precipitation assay, an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), an immunoblot assay (e.g., a western blot), an phosphorescence assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay. 
     
     
         8 . A method for identifying an individual having anti-influenza antibodies, comprising:
 a. contacting at least a portion of a sample from an individual with a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to at least one epitope from an influenza hemagglutinin (HA) protein such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the at least a portion of the sample and the nanoparticle are contacted under conditions suitable for forming a complex between the nanoparticle and the anti-influenza virus antibodies, if present;   b. analyzing the contacted sample for the presence of a nanoparticle/antibody complex, wherein the presence of such a complex indicates the individual has anti-influenza antibodies.   
     
     
         9 . The method of  claim 8 , wherein the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin and a mammalian ferritin. 
     
     
         10 . The nanoparticle of  claim 8 , wherein the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, where in the fusion protein is capable of self-assembling into nanoparticles. 
     
     
         11 . The nanoparticle of  claim 8 , wherein the hemagglutinin protein is from an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B). 
     
     
         12 . The nanoparticle of  claim 8 , wherein the fusion protein comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus. 
     
     
         13 . The nanoparticle of  claim 8 , wherein the nanoparticle comprises a second fusion protein comprising a second influenza hemagglutinin protein, wherein the first and second influenza hemagglutinin proteins are from different types, sub-types or strains of influenza viruses. 
     
     
         14 . The method of  claim 8 , wherein the step of analyzing comprises an assay selected from the group consisting of a hemagglutinin inhibition assay, an immunodiffusion assay, an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a precipitation assay, an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), an immunoblot assay (e.g., a western blot), an phosphorescence assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay. 
     
     
         15 . A method for measuring the response of an individual to a vaccine, comprising:
 a. administering to the individual a vaccine for influenza virus;   b. contacting at least a portion of a sample from the individual with a nanoparticle comprising a fusion protein, wherein the fusion protein comprises at least 25 contiguous amino acids from a monomeric ferritin subunit protein joined to at least one epitope from an influenza hemagglutinin (HA) protein such that the nanoparticle comprises trimers of the influenza virus HA protein epitope on its surface, and wherein the at least a portion of the sample and the nanoparticle are contacted under conditions suitable for forming a complex between the nanoparticle and the anti-influenza virus antibodies, if present; and,   c. determining the level of antibody present in the sample by determining the level of nanoparticle/antibody complex;   wherein an increase in the level of antibody in the sample over the pre-vaccination level of antibody indicates the vaccine induced an immune response in the individual.   
     
     
         16 . The method of  claim 15 , wherein the monomeric ferritin subunit protein is selected from the group consisting of a bacterial ferritin, a plant ferritin, an algal ferritin and a mammalian ferritin. 
     
     
         17 . The nanoparticle of  claim 15 , wherein the monomeric ferritin subunit protein comprises at least 25 contiguous amino acids an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, where in the fusion protein is capable of self-assembling into nanoparticles. 
     
     
         18 . The nanoparticle of  claim 15 , wherein the hemagglutinin protein is from an influenza virus selected from the group consisting of A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008 Bris, B). 
     
     
         19 . The nanoparticle of  claim 15 , wherein the fusion protein comprises an amino acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the nanoparticle elicits an immune response against an influenza virus. 
     
     
         20 . The method of  claim 15 , wherein the step of analyzing comprises an assay selected from the group consisting of a hemagglutinin inhibition assay, an immunodiffusion assay, an enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, a precipitation assay, an immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg, Switzerland), an immunoblot assay (e.g., a western blot), an phosphorescence assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, and an electronic sensory assay.