WO2014145245A2 - Methods and compositions for norovirus blockade epitopes - Google Patents

Methods and compositions for norovirus blockade epitopes Download PDF

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WO2014145245A2
WO2014145245A2 PCT/US2014/029969 US2014029969W WO2014145245A2 WO 2014145245 A2 WO2014145245 A2 WO 2014145245A2 US 2014029969 W US2014029969 W US 2014029969W WO 2014145245 A2 WO2014145245 A2 WO 2014145245A2
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gii
norovirus
epitope
capsid protein
chimeric
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PCT/US2014/029969
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French (fr)
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WO2014145245A3 (en
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Ralph Steven BARIC
Lisa Chon LINDESMITH
Kari Moore DEBBINK
Eric Francis DONALDSON
Jesica Anne SWANSTROM
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The University Of North Carolina At Chapel Hill
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Priority to EP14764479.3A priority Critical patent/EP2970395A4/en
Publication of WO2014145245A2 publication Critical patent/WO2014145245A2/en
Publication of WO2014145245A3 publication Critical patent/WO2014145245A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to compositions comprising norovirus blockade epitopes and methods of their use in therapeutic and diagnostic applications.
  • Noroviruses are the leading cause of severe viral gastroenteritis and are responsible for 50% of all acute gastroenteritis outbreaks in the United States and Europe [1], Although the severity of disease is usually moderate, lasting 1-3 days, infection can be especially virulent in young children, the elderly, and the immunocompromised, with the latter group experiencing chronic diarrhea and vims shedding for over a year [2-8], Importantly, it is estimated that 200,000 people die each year from norovirus infections, primarily children in the developing world [9]. An effective vaccine would be particularly advantageous for the very young and aged populations, military personnel, children and healthcare providers, food handlers, cruise ship passengers, and populations of the developing world [10],
  • Immunotherapeutics are especially needed for treating immuno suppressed populations experiencing long-term infections with chronic diarrhea.
  • the lack of understanding of the extensive antigenic relationships among the large number of norovirus strains and the complex relationship between host protective immunity and virus antigenic heterogeneity are the primary obstacles to norovirus vaccine development.
  • the present invention overcomes previous shortcoming in the art by providing norovirus blockade epitopes and methods of their use in therapeutic and diagnostic applications.
  • the present invention provides a chimeric norovirus capsid protein comprising a norovirus VP1 major capsid protein backbone comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the norovirus epitope and/or motif is from a norovirus strain that is different from the norovirus VP 1 major capsid protein backbone.
  • a synthetic backbone molecule comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the backbone molecule allows for formation of a conformational epitope and/or motif.
  • a norovirus P particle comprising a norovirus epitope and/or selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the epitope and/or motif is presented on the P particle surface.
  • the present invention provides a mimitope comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above.
  • the present invention further provides therapeutic methods.
  • therapeutic methods in one
  • the present invention provides a method of producing an immune response to a norovirus in a subject, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention.
  • Also provided herein is a method of treating a norovirus infection in a subject in need thereof, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention.
  • a method of preventing a disease or disorder caused by a norovirus infection in a subject comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention.
  • the present invention provides a method of protecting a subject from the effects of norovirus infection, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention.
  • the present invention also provides diagnostic methods.
  • the present invention provides a method of detecting a neutralizing antibody to a norovirus, the method comprising determining whether an antibody binds to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention, wherein binding by the antibody to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention indicates that the antibody is a neutralizing antibody to a norovirus.
  • a method is also provided herein of identifying a neutralizing antibody to a norovirus, comprising: (a) contacting an antibody with the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention; and (b) determining if the antibody binds to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention, wherein binding by the antibody to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention identifies the antibody as a neutralizing antibody to a norovirus.
  • the present invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a norovirus in a subject, the method comprising: (a) contacting a biological sample from a subject that has been administered the immunogenic composition with the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention;
  • Also provided herein is a method of identifying an immunogenic composition that induces a neutralizing antibody to a norovirus in a subject, the method comprising: (a) administering an immunogenic composition comprising a norovirus antigen to a subject in an amount effective to induce antibodies against the norovirus antigen; (b) contacting a biological sample from the subject with the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention;
  • FIG. 1 depicts the evolutionary profile of epitopes of various human norovirus (NoV) strains.
  • NoV human norovirus
  • the evolution of the GII.4 noro viruses was assessed over a 36 year period of time by comparing strains from 1 74 to 2010.
  • sites of variation in the p2 subdomain were noted, and these sites were mapped onto the crystal structure of the P- domain dirner for the 1997 strain VA387, Surface-exposed sites of variation were then examined to determine which residues were close enough to constitute a single epitope, and epitopes A-E and the NERK motif (referred to previously as Epitope F, this motif has been shown to influence binding of broadly neutralizing antibodies such as NVB 71.4 and
  • VLPs containing substituted Epitopes A through E and the NERK motif were created and tested for ability to bind to surrogate neutralizing antibodies in the VLP-HBGA blockade assay.
  • Epitopes A, D and E and the NERK motif were confirmed to be blockade epitopes.
  • Numbering of amino acids is based on amino acid position of the full amino acid sequence of the norovirus strains listed as provided in the GenBank ® Database or as provided herein.
  • the numbering of amino acids can be based on the amino acid sequence of
  • GII.4.2002 GenBank ® Accession No. AFJ04708.1 for amino acid sequence; GenBank ® Accession No. JQ478408 for nucleotide sequence, provided herein as SEQ ID NO:4.
  • amino acids of Epitope E are 406, 41 1 and 412.
  • the amino acids for Epitope E of the other listed norovirus strains are 407, 412, and 413 as shown, based on an insertion of an amino acid at position 394 in these subsequently listed strains.
  • the amino acid residues for Epitope D will be at alternative corresponding positions that would be readily recognized by one of ordinary skill in the art.
  • FIG. 2 depicts chimeric Venezuelan equine encephalitis replicon particles (VRPs) used to immunize mice.
  • VRPs Venezuelan equine encephalitis replicon particles
  • FIG. 3 depicts the immunization of mice with GII.4 parental or chimeric capsids.
  • FIG. 4 depicts blockade data with: (A) anti-2006.87A mouse sera; (B) anti- 2006.87A.02E mouse polyclonal sera; and (C) anti-2006-87A.02E mouse polyclonal sera on various VLPs.
  • FIG. 5 depicts blockade data with: (A) anti-1987 mouse sera; (B) anti-2002 mouse sera; (C) anti-2006 mouse sera; and (D) anti-2009 mouse sera on various VLPs.
  • FIG. 6 depicts the EC50 at which each sample reaches a blockade response for: (A) anti-GII .4-2006.87A mouse sera; (B) anti-GIL4-2006.87A.02E mouse sera; and (C) anti- Gil .4-2006-87/02/06 mouse sera with various virus like particles (VLPs).
  • VLPs virus like particles
  • FIG. 7 depicts the EC 50 at which each sample reaches a blockade response for: (A) anti-GII.4-1987 mouse sera; (B) anti-Gil 4-2002 mouse sera; (C) anti-GII.4-2006 mouse sera; and (D) anti-GII.4-2009 mouse sera with various VLPs.
  • FIG. 8 depicts the results of initial studies to identify a GII.4 norovirus conserved blockade epitope using human monoclonal antibodies.
  • FIG. 9 depicts that Epitope A comprises an evolving GIL4 blockade epitope recognized by NVB 114, 11 1 and 43.9,
  • FIG. 10 shows that residues 292, 295, and 373 are part of Epitope A.
  • FIG. 11 shows that amino acids 393-395 comprise a blockade epitope for
  • FIG. 12 Depicts that changes in amino acid position 391 alter Epitope D polar interactions, identifying 391 as a likely part of Epitope D
  • FIG. 13 depicts the characterization of Epitope E.
  • FIG. 14 depicts that the variation in Epitope E alters the structure of the caps id, identifying residues 355 and 357 as a likely part of Epitope E.
  • FIG. 15 depicts the expanded GII.4 blockade antibody epitopes over time.
  • FIG. 16 depicts the structural model of a conserved epitope.
  • FIG. 17 shows that ER motif substitutions decrease NVB 71.4 and GII.4.2002.G5 blockade potential with little impact on blockade temperature sensitivity.
  • FIG. 18 depicts the identification of residues that interact with the ERK motif.
  • FIG. 19 shows that residue 310 inversely modulates blockade potency and temperature sensitivity of the conserved GIL4 epitope.
  • FIG. 20 shows that chimeric VLPs induce a broadened blockade response.
  • FIG. 21 depicts a model of chimeric GII.4 VLP-based vaccine design
  • FIG. 22 depicts a method for the rational design of broadly cross-protective vaccines.
  • GII.4.1997 Panel A
  • GII.4.2006 Panel B
  • VLPs VLPs. Each symbol represents a different individual.
  • BD g0 values ⁇ 40 were scored as negative.
  • Total serum IgG binding to GII.4.1997 and GII.4.2006 was determined by enzyme immunoassay (EIA). Reciprocal EC 50 values are shown (right column Panel A and B). ⁇ Sera competing for binding of NVB 61.3; ⁇ Sera competing for binding of NVB 71.4; * Donor source of NVB 61.3 and 71.4.
  • FIG 24 Access of NVB 71.4 to the conserved GII.4 blockade epitope is temperature dependent. NVB 71.4 was assayed for ability to block the interaction of a panel of time-ordered GII.4 VLPs with carbohydrate ligand. Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment) o
  • FIG 25 Access of GII.4.2002.G5 to a conserved GII.4 blockade epitope is regulated by temperature. GII.4.2002.G5 was assayed for ability to block the interaction of
  • Non-blockade VLPs were assigned an EC 50 of 2X the upper limit of detection for statistical analysis and denoted by a data marker on the graph above the dashed line (assay upper limit of detection) for visual comparison. Error bars represent 95% confidence intervals.
  • FIG 26 Antibody access to the conserved epitope is not temperature sensitive on
  • GII.4.2009 VLPs made at lower temperature in insect cells.
  • Epitope A human mab Panel A
  • NVB 71.4 Panel B
  • GII.4.2002.G5 Panel C
  • Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatrnent compared to the amount of VLP bound in the absence of antibody pretreatment) and the mean EC 50 ( ⁇ / ⁇ ) titer for blockade calculated.
  • EC 50 titer was defined as the mean EC 50 at 37 C compared to room temperature. * Mean
  • FIG 27 Characterization of VLPs with substitutions in predicted conserved antibody epitopes.
  • Schematic of constructs Panel A. Particle integrity was verified by transmission electron microscope visualization (Panel B) and carbohydrate ligand (Pig Gastric Mucin type III, PGM) binding of VLPs (Panel C).
  • Non-PGM binding VLPs were assigned an EC 50 of 2X the upper limit of detection for statistical analysis and denoted by a data marker on the graph above the dashed line (assay upper limit of detection) for visual comparison. Error bars represent 95% confidence intervals.
  • FIG. 28 Characterization of VLPs with substitutions in residue 310 and ERK.
  • FIG. 29 Antibody-bound VLPs retain structural integrity.
  • GO.4.2009 Panel A
  • GII.4.2009.ERK Panel B
  • VLPs were immuno-stained with NVB 71.4 or epitope A (GII.4.2009 only, Panel C) human mabs and visualized by negative stain transmission electron microscopy. Arrows denote immuno-gold labeled VLPs.
  • FIG 30 Binding of NVB 71.4 does not disrupt surface epitope A topolo y.
  • Human mabs to surface epitope A or NVB 71.4 were evaluated for ability to block binding of mouse mabs to epitope A or the conserved blockade epitope in GII.4.1997 (Panel A) and GII.4.2006 (Panel B) using a BOB assay.
  • Sigmoidal curves were fit to the mean percent control binding (percent of mouse mab bound to VLP in the presence of human mab pretreatment compared to the amount of mouse mab bound in the absence of human mab pretreatment) and the mean EC 50 fag/ml) titer for blockade of binding calculated, R EC- 0 > 8 g ml, " EC 50 ⁇ 1 ⁇ / ⁇ 1.
  • FIG 31 The mapped epitopes of GII.4 noroviruses.
  • the previously described evolving antibody blockade epitopes A-E are shown on the surface next to the carbohydrate binding sites (circled) (Panel A).
  • the ERK motif is distal to the carbohydrate binding sites (denoted by arrows) (Panel B).
  • FIG. 32 Proposed model for regulation of antibody access to the conserved GII.4 blockade epitope/s by the ERK motif and VLP structural conformation.
  • Noroviruses are single-stranded, positive sense RNA viruses that belong to the family Caliciviridae.
  • the norovirus genome is ⁇ 7.5 kb and contains three open reading frames.
  • ORF1 encodes the non-structural proteins
  • ORF2 encodes the VPl major capsid protein
  • ORF3 encodes the VP2 minor capsid protein.
  • VPl is divided into three parts: the shell, which forms the irrner-most portion of the virion; the PI subdomain, which forms a staik-like projection away from the virion surface; and the P2 subdomain, which is a highly variable, surface exposed region that sits on top of PI .
  • the P2 subdomain of the VPl major capsid protein contains potential neutralizing antibody epitopes. These epitopes change over time, likely allowing new strains to escape human herd immunity.
  • the present invention is based on the identification and characterization of various neutralizing blockade epitopes in norovirus capsid protein, which has led, in some embodiments, to the development of a vaccine platform to allow a single immunogenic protein to protect against multiple strains of norovirus.
  • the present invention provides a chimeric norovirus capsid protein comprising a norovirus VPl major capsid protein backbone comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NER motif; and g) any combination of (a) through (f) above, wherein the norovirus epitope and/or motif is from a norovirus strain that is different from the norovirus ORF2 major capsid protein backbone.
  • Nonlimiting examples of Epitope A, Epitope B, Epitope C, Epitope D, Epitope E and the NERK motif are provided for several norovirus strains, as shown for example, in Figure 1 and Figure 15 herein.
  • a synthetic backbone molecule comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the backbone molecule allows for formation of the conformational epitope and/or motif.
  • a norovirus P particle comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the epitope and/or motif is presented on the P particle surface.
  • the present invention provides a mimitope comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above.
  • the norovirus VP1 major capsid protein backbone can be from any norovirus strain and in some embodiments can be from a genogroup Gil, genotype 4 (GII.4) strain.
  • the norovirus VP1 major capsid protein backbone can be from GII.4.1974 (GenBank ® Database Accession No. ACT76139.1), GII.4.1987 (GenBank ® Database Accession No.
  • amino acid sequence of GII.4.2002a is as follows (SEQ ID NO: 5).
  • the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope A (amino acids 294, 296, 297, 298, 368 and 372) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII
  • the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope B (amino acids 333 and 382) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GIIA2008a ⁇ GII.4.2009 or GII.4.2012.
  • the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope C (amino acids 340 and 376) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GO.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GIL4.2008a, GII.4.2009 or GII.4.2012.
  • the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope D (amino acids 3 3, 394 and 395) from GII.4.1 74, GII.4.1 87, GII.4.1997, GO ,4.2002a, GII.4.2002, GII.4.2004,
  • Epitope D is from a norovirus strain different from the norovirus ORF2 major capsid protein backbone.
  • the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope E (amino acids 407, 412 and 413) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope E is from a norovirus strain different from the norovirus ORF2 major capsid protein backbone.
  • the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise the NER motif (previously referred to as Epitope F) (amino acids 310, 316, 484 and 493) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012.
  • the NER motif previously referred to as Epitope F
  • the norovirus ORF2 major capsid protein backbone can be from GII.4.2006 and can comprise Epitope A from GII.4.1987 and Epitope E from GII.4.2002.
  • the present invention further provides an isolated nucleic acid molecule encoding the chimeric norovirus capsid protein of this invention, a vector comprising the nucleic acid molecule of this invention and a cell comprising the capsid protein, nucleic acid molecule and/or vector of this invention.
  • the present invention provides a Venezuelan Equine
  • VEE Encephalitis
  • VRP replicon particle
  • the present invention provides a virus like particle (VLP) comprising the chimeric norovirus capsid protein of this invention.
  • VLP virus like particle
  • the present invention also provides a composition comprising the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention and/or the VLP of this invention in a pharmaceutically acceptable carrier.
  • the individual and conformational epitopes of the norovirus capsid proteins can be presented on a synthetic backbone or support structure so that the epitopes within the synthetic backbone or support structure mimic the conformation and arrangement of the epitopes within the structure of the norovirus capsid protein, VLP or VRP.
  • the present invention provides peptide mimitopes (see, Meloen et al. (2000) J. Mol. Recognit, 13, 352-359) that mimic the individual and conformational epitopes of the norovirus capsid proteins of the invention.
  • Mimitopes may be identified using any technique known in the art, such as by surface stimulation, random peptide libraries or phage display libraries, using an antibody or antibodies to the individual and conformational epitopes of the chimeric norovirus capsid proteins of the invention.
  • a norovirus P particle ⁇ see, Tan et al. (2011) J Virol. 85(2), 753-764) is provided that presents the epitopes of the norovirus capsid proteins as described herein.
  • one or more of the identified potential neutralization epitopes from one or more donor strains is moved into any other Gil .4 noro virus backbone strain to induce broad protection against multiple strains.
  • moving epitope A from GII.4.1987 and epitope E from GII.4.2002 into the GII.4.2006 backbone induces a broadly blocking immune response in mice against GII.4 strains GII.4.1987, 2002, 2006, 2009, and 2012.
  • a vaccine that incorporates epitope(s) from one or more circulating strains into the backbone of another strain can induce protection against multiple norovirus strains.
  • the full-length ORF2 major capsid gene sequence from norovirus is either cloned from a patient sample or produced as a synthetic construct (e.g., from a commercial source). Natural or engineered endonuclease sites are used to insert sequence containing the desired epitope changes for one or more GII.4 strains.
  • the full-length capsid may be synthesized (e.g., using a consensus sequence) with the desired sequence changes already present.
  • this gene is then cloned into an expression vector, Upon expression, the VPl major capsid protein self-assembles into VLPs, which can then be purified.
  • VRPs expressing the major capsid protein can be produced and purified and subsequently used as a vaccine or used as a source of VLP production.
  • chimeric norovirus capsid protein and similar terms will be understood in the art to mean a norovirus capsid protein derived from a particular norovirus strain that contains single or multiple amino acid substitutions at various positions in which the amino acid substitution(s) is an amino acid(s) that is one from the corresponding position(s) of a norovirus capsid protein from a different norovirus strain.
  • the amino acid substitution comprises a particular epitope from a norovirus strain different from that of the capsid protein in which the substitution is made.
  • the amino acid substitutions may be at amino acids 292, 294, 295, 296, 297, 298, 357, 365, 368, 372 and 373 (Epitope A) of the norovirus VPl major capsid protein encoded by open reading frame 2 (ORF2) of human norovirus (hNoV),
  • the substitutions may be at amino acids 333 and 382 (Epitope B) of the hNoV VPl major capsid protein.
  • the substitutions may be at amino acids 340 and 376 (Epitope C) of the hNoV VPl major capsid protein.
  • the substitutions may be at amino acids 391, 393, 394, 395, 396 and 397 (Epitope D) of the hNoV VPl major capsid protein. In some embodiments, the substitutions may be at amino acids 355, 356,357, 359, 364, 365, 404, 407, 412 and 413, or the corresponding amino acids (e.g., 406, 41 1 and 412) of certain NoV strains, (Epitope E) of the hNoV VP1 major capsid protein. In still further,
  • the amino acid substitutions may be at amino acid positions 310, 316, 484 and 493 (NERK motif, previously referred to as Epitope F) of the hNoV VP1 major capsid protein.
  • the amino acid substitutions may comprise any two or more (e.g., 2, 3, 4, or 5) of these epitopes and/or motifs, in any combination in the same norovirus capsid protein.
  • Nonlimiting examples of the epitopes and motifs of this invention that can be combined in the same norovirus capsid protein include the following combinations (wherein A is Epitope A, B is Epitope B, C is Epitope C, D is Epitope D, E is Epitope E and F is the NERK motif): AB, AC, AD, AE, AF, BC, BD, BE, BF, CD, CE, CF, DE, DF, EF, ABC, ABD, ABE, ABF, ACD, ACE, ACF, ADE, ADF, AEF, ABCD, ADEF, ABEF, ACEF, ABCE, ABCF, ABDF, ABCE, ACDF, BCDEF, BCDE, BCD, CDEF, CDE, CDF, DEF, A
  • the amino acid residues of a single epitope or motif(e.g., Epitope A, Epitope B, Epitope C, Epitope D, Epitope E, NERK motif) from a GII.4 norovirus strain is substituted into a capsid protein backbone of a different GII.4 norovirus strain.
  • the present invention is based on the discovery and
  • GII.4 capsid protein that function as neutralization epitopes (i.e., blockade epitopes) and motifs that induce a protective immune response to GII.4 norovirus strains.
  • the identification of the amino acid residues that make up these epitopes and motifs can be applied in developing a vaccine platform, wherein one or more of these epitopes and/or motifs from one or more donor norovirus strains are introduced into a different GII.4 norovirus capsid protein backbone to produce a chimeric norovirus capsid protein that induces broad protection against multiple norovirus strains.
  • amino acid residue at a given position in the norovirus capsid protein can be any amino acid residue that makes up a blockade epitope or motif.
  • amino acid residue 365 has been identified to be either I or V in the respective GII.4 norovirus strains listed in Figure 15.
  • an I or V substitution can be made in a norovirus capsid protein backbone, in addition to substitutions at the other amino acid residue sites that make up Epitope A, such that the capsid protein backbone comprises all of the amino acid residues that make up Epitope A of a particular GII.4 strain that is different from the backbone strain.
  • uncharacterized GII.4 norovirus strain is found to have an amino acid residue at position 365 that is not I or V, such newly identified amino acid residue can be substituted into a norovirus capsid protein backbone at position 365, along with all of the other amino acid residues identified in the emerging or previously uncharacterized norovirus strain at the other amino acid positions that make up Epitope A, in order to produce a chimeric norovirus capsid protein of this invention that can induce an immunoprotective response against not only the emerging or previously uncharacterized GII.4 norovirus strain, but also against other GII.4 norovirus strains.
  • the present invention provides for the production of chimeric GII.4 capsid proteins as described herein that can be used to immunize subjects against a newly emerging GII.4 strain as soon as the amino acid residues of a blockade epitope or motif of this invention are identified in the new strain.
  • the term "norovirus capsid protein backbone” and similar terms refer to the particular norovirus capsid protein from which a chimeric norovirus capsid protein is based.
  • the norovirus capsid protein backbone may be from any geno group, genotype and strain of hNoV.
  • the norovirus capsid protein backbone is from genogroup II and genotype 4 (GII.4) of hNoV.
  • the norovirus capsid protein backbone may be from GII.4-1974 (GenBank Access. No. ACT76139.1).
  • the norovirus capsid protein backbone may be from GO.4-1 87 (GenBank Access. No. AA 50355.1).
  • the norovirus capsid protein backbone may be from GII.4-1997 (GenBank Access. No. AFJ04707.1 ). In another embodiment, the norovirus capsid protein backbone may be from GO .4-2002 (GenBank Access. No.
  • the norovirus capsid protein backbone may be from GII.4-2004 (GenBank Access. No. AAZ31376.2). In another embodiment, the norovirus capsid protein backbone may be from GII.4-2005 (GenBank Access. No, BAE98194.1). In another embodiment, the norovirus capsid protein backbone may be from GO.4-2006 (GenBank Access. No. AFJ4709.1). In another embodiment, the norovirus capsid protein backbone may be from GII.4-2007 (GenBank Access. No. BAH56690.1), In another embodiment, the norovirus capsid protein backbone may be from GII.4-2008a (GenBank Access. No. ACX31885.1).
  • the norovirus capsid protein backbone may be from GO.4-2008s (GenBank Access. No. BAH30307.1). In another embodiment, the norovirus capsid protein backbone may be from GII.4-2009 (GenBank Access. No.
  • the norovirus capsid protein backbone may be from GIL4-2012 (GenBank Access. No. AFV08795.1).
  • the epitope of the chimeric norovirus capsid protein may be from any norovirus genogroup, genotype and strain, as long as it different from that of the norovirus capsid protein backbone.
  • the norovirus capsid protein backbone is from GII.4-2006
  • the epitope is epitope A from GII.4-1987.
  • the norovirus capsid protein backbone is from GII.4-2006
  • the epitopes can be a combination of Epitope A from GIL 1987 and Epitope E from GII.4-2002.
  • the invention further provides a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding a chimeric norovirus capsid protein or a polypeptide of the invention.
  • a nucleic acid molecule e.g., an isolated nucleic acid molecule
  • the invention further provides a nucleic acid molecule (e.g. , an isolated nucleic acid molecule) encoding a chimeric norovirus VLP, a chimeric norovirus VRP or a viral coat of a chimeric norovirus particle of the invention.
  • a nucleic acid molecule e.g. , an isolated nucleic acid molecule
  • vectors encoding the nucleic acid molecules of the invention.
  • cells that comprise the vectors, nucleic acid molecules, norovirus epitopes, polypeptides, chimeric norovirus VLPs, chimeric norovirus VRPs and/or chimeric norovirus particles of the invention.
  • the invention also provides immunogenic compositions comprising the cells, vectors, nucleic acid molecules, norovirus epitopes, chimeric norovirus capsid proteins, polypeptides, chimeric norovirus VLPs, chimeric norovirus VRPs, and/or chimeric norovirus particles of the invention in a pharmaceutically acceptable carrier.
  • the immunogenic composition is monovalent. In some embodiments, the immunogenic composition is multivalent for different norovirus serotypes.
  • the invention encompasses methods of producing an immune response to a norovirus in a subject, the method comprising administering to the subject an effective amount of a norovirus epitope, a chimeric norovirus capsid protein, a polypeptide, a chimeric norovirus VLP, chimeric norovirus VR1P or chimeric norovirus particle, nucleic acid molecule, vector, cell and/or immunogenic composition of the invention.
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel character! stic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 21 11.03. Thus, the term “consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising.”
  • a measurable value such as an amount or concentration (e.g., the amount of a substrate)
  • a measurable value such as an amount or concentration (e.g., the amount of a substrate)
  • "about X" where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measureable value may include any other range and/or individual value therein.
  • nucleic acid encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g. , chemically synthesized) DNA and chimeras of RNA and DNA.
  • the nucleic acid may be double-stranded or single-stranded.
  • the nucleic acid may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • polypeptide encompasses both peptides and proteins
  • a “fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame.
  • a "recombinant" nucleic acid, polynucleotide or nucleotide sequence is one produced by genetic engineering techniques.
  • a "recombinant" polypeptide is produced from a recombinant nucleic acid, polypeptide or nucleotide sequence.
  • an “isolated” polynucleotide e.g., an “isolated nucleic acid” or an “isolated nucleotide sequence
  • an isolated polynucleotide means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the "isolated" e.g., an "isolated nucleic acid” or an “isolated nucleotide sequence
  • polynucleotide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration).
  • the isolated polynucleotide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
  • an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • the "isolated" polypeptide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five -hundred -fold, one thousand-fold, ten thousandfold or greater concentration).
  • the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
  • an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier.
  • immunogen and "antigen” are used interchangeably herein and mean any compound (including polypeptides) to which a cellular and/or humoral immune response can be directed.
  • an immunogen or antigen can induce a protective immune response against the effects of noro irus infection.
  • Effective amount refers to an amount of a vector, nucleic acid molecule, epitope, polypeptide, cell, composition or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect.
  • the effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art, As appropriate, an "effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
  • immunogenic amount or "effective immunizing dose,” as used herein, unless otherwise indicated, means an amount or dose sufficient to induce an immune response (which can optionally be a protective response) in the treated subject that is greater than the inherent immunity of non-immunized subjects.
  • An immunogenic amount or effective immunizing dose in any particular context can be routinely determined using methods known in the art.
  • vaccine means vacuna
  • vaccination means a process or composition that increases a subject's immune reaction to an immunogen (e.g. , by providing an active immune response), and therefore its ability to resist, overcome and/or recover from infection (i.e., a protective immune response).
  • treat By the terms “treat,” “treating” or “treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • the term “treat,” “treating” or “treatment of (and grammatical variations thereof) refer to a reduction in the severity of viremia and/or a delay in the progression of viremia, with or without other signs of clinical disease.
  • a “treatment effective” amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • prevent refers to prevention of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject relative to what would occur in the absence of the methods of the invention.
  • the terms “prevent,” “preventing” or “prevention of (and grammatical variations thereof) refer to prevention of viremia in the subject, with or without other signs of clinical disease.
  • the prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s).
  • the prevention can also be partial, such that the occurrence of the disease, disorder and or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the methods of the present invention.
  • a "prevention effective” amount as used herein is an amount that is sufficient to prevent (as defined herein) the disease, disorder and/or clinical symptom in the subject.
  • the efficacy of treating and/or preventing a disease or disorder caused by norovirus infection by the methods of the present invention can be determined by detecting a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters (e.g., viremia), as would be well known to one of skill in the art.
  • a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters (e.g., viremia), as would be well known to one of skill in the art.
  • the terms "protect,” “protecting,” “protection” and “protective” encompass both methods of preventing and treating norovirus infection in a subject, whether against one or multiple strains, genotypes or genogroups of norovirus.
  • protective immune response or “protective” immunity indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence and/or severity and/or duration of disease or any other manifestation of infection.
  • a protective immune response or protective immunity results in reduced viremia, whether or not accompanied by clinical disease.
  • a protective immune response or protective immunity may be useful in the therapeutic treatment of existing disease.
  • an “active immune response” or “active immunity” is characterized by “participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both.” Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 1 17 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination.
  • Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host," Id.
  • methods are provided of treating a norovirus infection in a subject in need thereof by passive immunity, comprising
  • an antibody that specifically reacts with a norovirus e.g., an antibody that binds a blockade epitope or motif of a GII.4 norovirus.
  • a norovirus e.g., an antibody that binds a blockade epitope or motif of a GII.4 norovirus.
  • GII.4.2002.G5. It would be understood that if the subject to be treated is a human, a humanized mouse monoclonal antibody (e.g., humanized GII.4.2002.G5) would be administered to the subject.
  • a humanized mouse monoclonal antibody e.g., humanized GII.4.2002.G5
  • the production of humanized monoclonal antibodies from non- human species is well known in the art, as are protocols for determining dosage and route of administration of such antibodies to a subject in an amount effective to treat a norovirus infection and/or protect a subject against the effects of norovirus infection.
  • a "subject" of the invention includes any animal susceptible to norovirus infection.
  • a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.).
  • the subject is a primate subject, a non-human primate subject (e.g. , a chimpanzee, baboon, monkey, gorilla, etc) or a human.
  • Subjects of the invention can be a subject known or believed to be at risk of infection by norovirus.
  • a subject according to the invention can also include a subject not previously known or suspected to be infected by norovirus or in need of treatment for norovirus infection.
  • Subjects may be treated for any purpose, such as for eliciting a protective immune response or for eliciting the production of antibodies in that subject, which antibodies can be collected and used for other purposes such as research or diagnostic purposes or for administering to other subjects to produce passive immunity therein, etc.
  • Subjects include males and/or females of any age, including neonates, juvenile, mature and geriatric subjects.
  • the subject can be an infant (e.g. , less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or younger), a toddler (e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months), or a child (e.g., at least about 1, 2, 3, 4 or 5 years of age and/or less than about 14, 12, 10, 8, 7, 6, 5, or 4 years of age).
  • the subject is a human subject that is from about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 12 to 18, 24, 36, 48 or 60 months of age, from about 18 to 24, 30, 36, 48 or 60 months of age, or from about 24 to 30, 36, 48 or 60 months of age.
  • a "subject in need" of the methods of the invention can be a subject known to be, or suspected of being, infected with, or at risk of being infected with, noro virus.
  • compositions comprising the norovirus epitopes, chimeric norovirus capsid proteins, polypeptides, chimeric norovirus VLPs, chimeric norovirus VRPs or chimeric norovirus particles, nucleic acids, vectors, cells or compositions of the invention and a pharmaceutically acceptable carrier are also provided, and can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (latest edition), in the manufacture of a pharmaceutical composition according to embodiments of the present invention, the composition of the invention is typically admixed with, inter alia, a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant a carrier that is compatible with other ingredients in the pharmaceutical composition and that is not harmful or deleterious to the subject.
  • the carrier may be a solid or a Hquid, or both, and is preferably formulated with the composition of the invention as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the composition.
  • the pharmaceutical compositions are prepared by any of the well- known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients.
  • the pharmaceutically acceptable carrier is sterile and would be deemed suitable for administration into human subjects according to regulatory guidelines for pharmaceutical compositions comprising the carrier.
  • a "pharmaceutically acceptable” component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition.
  • Non-limiting examples of pharmaceutically acceptable components include any of the standard
  • compositions such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents, typically provided as sterile compositions.
  • pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents, typically provided as sterile compositions.
  • the compositions of the invention can further comprise one or more than one adjuvant.
  • the adjuvants of the present invention can be in the form of an amino acid sequence, and/or in the form or a nucleic acid encoding an adjuvant.
  • the adjuvant can be a component of a nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) and/or a separate component of the composition comprising the nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) of the invention.
  • the adjuvant can also be an amino acid sequence that is a peptide, a protein fragment or a whole protein that functions as an adjuvant, and/or the adjuvant can be a nucleic acid encoding a peptide, protein fragment or whole protein that functions as an adjuvant.
  • adjuvant describes a substance, which can be any immunomodulating substance capable of being combined with a composition of the invention to enhance, improve or otherwise modulate an immune response in a subject.
  • the adjuvant can be, but is not limited to, an
  • immunostimulatory cytokine including, but not limited to, GM CSF, interleukin-2, interleukin-12, interferon-gamma, interIeukin-4, tumor necrosis factor-alpha, mterleukin- 1 , hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules
  • SYNTEX adjuvant formulation 1 SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
  • Suitable adjuvants also include an aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes.
  • aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin
  • alum aluminum hydroxide gel
  • aluminum phosphate aluminum phosphate
  • algannmulin algannmulin
  • adjuvants are well known in the art and include without limitation MF 59, LT- K63, LT-R72 (Pal et al., Vaccine 24(6):766-75 (2005)), QS-21 , Freund's adjuvant (complete and incomplete), aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn - glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryi lipid
  • Additional adjuvants can include, for example, a combination of monophosphoryi lipid A, preferably 3-de-O-acylated monophosphoryi. lipid A (3D-MPL) together with an aluminum salt.
  • An enhanced adjuvant system involves the combination of a monophosphoryi lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in PCT publication number WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in PCT publication number WO
  • nucleic acid compositions of the invention can include an adjuvant by comprising a nucleotide sequence encoding the antigen and a nucleotide sequence that provides an adjuvant function, such as CpG sequences.
  • CpG sequences, or motifs are well known in the art.
  • an adjuvant for use with the present invention such as, for example, an adjuvant for use with the present invention, such as, for example, an adjuvant for use with the present invention
  • immunostimulatory cytokine can be administered before, concurrent with, and/or within a few hours, several hours, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 days before and/or after the administration of a composition of the invention to a subject.
  • any combination of adjuvants such as immunostimulatory cytokines, can be co-administered to the subject before, after and/or concurrent with the administration of an immunogenic composition of the invention.
  • adjuvants such as immunostimulatory cytokines
  • immunostimulatory cytokines can consist of two or more immunostimulatory cytokines, such as GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules.
  • the effectiveness of an adjuvant or combination of adjuvants can be determined by measuring the immune response produced in response to administration of a composition of this invention to a subject with and without the adjuvant or combination of adjuvants, using standard procedures, as described herein and as known in the art.
  • the adjuvant comprises an alphavirus adjuvant as described, for example in U.S. 7,862,829.
  • Boosting dosages can further be administered over a time course of days, weeks, months or years. In chronic infection, initial high doses followed by boosting doses may be advantageous.
  • the pharmaceutical formulations of the invention can optionally comprise other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, diluents, salts, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanol amine oleate, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and is typically in a solid or liquid particulate form.
  • compositions of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (9 th Ed. 1995).
  • the VLPs are typically admixed with, inter alia, an acceptable carrier.
  • the carrier can be a solid or a liquid, or both, and is optionally formulated with the compound as a unit-dose formulation, for example, a tablet.
  • aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N J.), and the like.
  • aqueous carriers e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N J.), and the like.
  • These compositions can be sterilized by conventional techniques.
  • the formulations of the invention can be prepared by any of the well-known techniques of pharmacy.
  • the pharmaceutical formulations can be packaged for use as is, or lyophilized, the lyophilized preparation generally being combined with a sterile aqueous solution prior to administration.
  • the compositions can further be packaged in unit/dose or multi-dose containers, for example, in sealed ampoules and vials.
  • the pharmaceutical formulations can be formulated for administration by any method known in the art according to conventional techniques of pharmacy.
  • the compositions can be formulated to be administered intranasally, by inhalation (e.g., oral inhalation), orally, buccally (e.g. , sublingually), rectally, vaginally, topically, intrathecal ly, intraocularly, transdermally, by parenteral administration (e.g.
  • intramuscular e.g. , skeletal muscle
  • intravenous subcutaneous, intradermal, intrapleural, intracerebral and intra-arterial, intrathecal
  • topically e.g., to both skin and mucosal surfaces, including airway surfaces.
  • the pharmaceutical formulation can be formulated as an aerosol (this term including both liquid and dry powder aerosols).
  • the pharmaceutical formulation can be provided in a finely divided form along with a surfactant and propellant Typical percentages of the composition are 0.01-20% by weight, preferably 1-10%.
  • the surfactant is generally nontoxic and soluble in the propellant.
  • esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, if desired, as with lecithin for intranasal delivery.
  • Aerosols of liquid particles can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. Intranasal administration can also be by droplet administration to a nasal surface.
  • Injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one can administer the pharmaceutical formulations in a local rather than systemic manner, for example, in a depot or sustained-release formulation.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile formulation of the invention in a unit dosage form in a sealed container can be provided.
  • the formulation can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject.
  • the unit dosage form can be from about 1 ⁇ to about 10 grams of the formulation.
  • a sufficient amount of emulsifying agent which is pharmaceutically acceptable, can be included in sufficient quantity to emulsify the formulation in an aqueous carrier.
  • emulsifying agent is phosphatidyl choline.
  • compositions suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tables, as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water- in-oil emulsion.
  • Oral delivery can be performed by complexing a compound(s) of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art.
  • Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the protein(s) and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • the pharmaceutical formulations are prepared by uniformly and intimately admixing the compound(s) with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet can be prepared by compressing or molding a powder or granules, optionally with one or more accessory ingredients.
  • Compressed tablets are prepared by compressing, in a suitable machine, the formulation in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets are made by molding, in a suitable machine, the powdered protein moistened with an inert liquid binder.
  • compositions suitable for buccal (sub-lingual) administration include lozenges comprising the compound(s) in a flavored base, usually sucrose and acacia or tragacanth; and pastilles in an inert base such as gelatin and glycerin or sucrose and acacia.
  • compositions suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacterio stats and solutes, which render the composition isotonic with the blood of the intended recipient.
  • Aqueous and non- aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents.
  • non -aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions suitable for rectal administration are optionally presented as unit dose suppositories. These can be prepared by admixing the active agent with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.
  • compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers that can be used include, but are not limited to, petroleum jelly, lano ine, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • topical delivery can be performed by mixing a pharmaceutical formulation of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
  • a lipophilic reagent e.g., DMSO
  • compositions suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time.
  • Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3:318 (1986)) and typically take the form of a buffered aqueous solution of the compound(s).
  • Suitable formulations can comprise citrate or bisNtris buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M active ingredient.
  • composition can be formulated as a liposomal formulation.
  • the lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free.
  • the liposomes that are produced can be reduced in size, for example, through the use of standard sonication and homogenization techniques.
  • the liposomal formulations can be Iyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • the immunogenic formulations of the invention can optionally be sterile, and can further be provided in a closed pathogen-impermeable container.
  • the dosage of a vims particle of this invention can be in a range of about 10 to about 10 plaque forming units (PFUs),
  • the dosage of a VLP of this invention can be in a range of about 5 micrograms to 5 milligrams.
  • the dosage of a protein of this invention can be in a range of about 10 to about 10 5 micrograms +/- adjuvant.
  • Noroviruses are ⁇ 38 nm icosahedral viruses with a ⁇ 7.5 Kb single-stranded, positive- sense RNA genome that encodes three large open reading frames (ORFs).
  • ORF1 encodes the non-structural proteins
  • ORFs 2 and 3 encode the major and minor capsid proteins, respectively.
  • Expression of the major capsid protein (ORF2) in Venezuelan equine encephalitis (VEE) virus or baculovirus results in the formation of virus-like particles (VLPs) composed of 90 copies of the major capsid protein dimer [11].
  • Noroviruses are grouped by the amino acid sequence of the major capsid protein.
  • Viruses with less than 14.3% difference are classified as the same strain, 14,3-43.8% the same genotype, and 45-61.4% the same genogroup [12].
  • noroviruses are grouped into five genogroups (GI-GV).
  • Genogroups GI and GII are responsible for most human infections and are further subdivided into 8 and 21 different genotypes, respectively [1, 12].
  • the capsid monomer is divided into three domains.
  • the shell domain (S) forms the core of the particle and the protruding domain (P) extends away from the core.
  • the P domain is further subdivided into the PI subdomain (residues 226-278 and 406-520) and the P2 subdomain (residues 279-405) [1 1].
  • the P2 subdomain is the most exposed region of the viral particle and it is well positioned to interact with potential neutralizing antibodies and histoblood group antigen (HBGA) ligands [13-17].
  • HBGA histoblood group antigen
  • Epitope A encodes significant amino acid changes over time and has also been demonstrated to be an evolving GII.4 blockade epitope using mouse mAbs [42].
  • Epitope A is conformational and is located on the top of the capsid proximal to the HBGA binding pocket. Six variable sites were close to each other in the region of this putative epitope, suggesting that these residues may work in concert to change the local structure of Epitope A.
  • the variable, surface-exposed residues include amino acid positions: 294, 296-298, 368 and 372. In the present invention, additional residues at amino acid positions 292, 295, 357, 365, and 373 have been identified as residues of Epitope A.
  • Epitope A is continuing to evolve in extant strains, whereby the amino acid at position 294 seems to vary extensively in strains from 2008-2010 (amino acid replacements P294A, P294S and P294T have been observed at this position).
  • Epitope B was identified based upon two variable residues at positions 333 and 382. While these residues are buried in the dimer interface between two chains, the patterns of variation at these sites suggest that they play an important role in the evolution of novel strains, perhaps by evolving replacements that allow the more surface exposed residues in other surface exposed epitopes to dramatically change the physiochemical properties of the amino acid replacements.
  • Residues 340 and 376 make up the variable residues of putative Epitope C.
  • This putative conformation dependent epitope is on the surface and lateral edge of the capsid and is directly proximal to the HBGA binding pocket, suggesting that this epitope may play a role in receptor switching along with Epitope D.
  • Epitope D was initially characterized as comprising the three variable residues from positions 393-395. In the present invention, Epitope D has been identified as further comprising amino acid residues at positions 391, 396 and 397. In the first reported crystal structure for the GII.4 noroviruses, this region was reported to be a secondary HBGA binding site [16]. However, the location of this epitope on the surface of the capsid, directly proximal to the HBGA binding site, suggests that it likely plays a role in both receptor switching and in escape from herd immunity and perhaps both, simultaneously [13, 21, 43, 44]. Epitope D is close enough to the HBGA binding pocket to contribute to or inhibit carbohydrate binding, and yet variable enough to suggest that it is targeted by the immune response.
  • Putative Epitope E comprises variable residues 407, 412 and 413, which are surface exposed regions lateral to the HBGA binding pockets and other epitopes.
  • residues at amino acid positions 355, 356, 357, 359, 364, 365 and 404 have been identified as being included in Epitope E.
  • the residues 407, 412 and 413 vary with every major epidemic strain after 2002, suggesting that it is a hot spot for the emergence of immunologically novel GII.4 strains.
  • Epitope E is a GIL4.2002 blockade antibody epitope [45], The fact that this putative epitope is lateral to the HBGA binding pockets suggests that antibodies are targeting regions interior to the capsid surface, which suggests that other epitopes may be present in the PI subdomain. A few variable residues do not necessarily identify the boundaries of a putative epitope. Moreover, it is nearly impossible to predict the surface area of a putative epitope by sequence analysis alone.
  • Epitope A comprises amino acids 292, 294, 295, 296, 297, 298, 357, 365, 368, 372 and 373 and is a highly variable blockade epitope that changes with new GII.4 strain emergence.
  • Monoclonal human and mouse antibodies against Epitope A have been described as highly strain selective, lacking reactivity to other GIL4 strains chronologically removed from the immunizing strain.
  • GII.4.2006 and GII.4.2009 were found to differ in reactivity to a human mAb targeting Epitope A, establishing a difference in human neutralizing epitopes between these two strains and supporting escape from herd immunity by antigenic variation at neutralizing epitopes as a mechanism for new NoV strain emergence.
  • Epitope D (residues 391, 393, 394, 395, 396 and 397) has also been confirmed as an evolving blockade epitope using human anti-No V mAbs. These findings are particularly interesting as Epitope D has been shown to modulate HBGA binding of GII.4 strains supporting the suggested correlation between epitope escape from herd immunity and altered HBGA binding [13 ].
  • Table 1 lists several NoV strains of this invention.
  • the evolutionary profile of the epitopes of various human NoV strains is shown in FIG 1.
  • Five important antigenic epitopes in GII.4 noroviruses were identified.
  • the differences between the epitope amino acid sequences in GII.4-1987 and GIL4-2006 are shown in Table 2.
  • Three of these, epitopes A, D, and E, have been confirmed as GII.4 blockade epitopes.
  • Epitope A is at positions 292, 294, 295, 296, 297, 298, 357, 365, 368, 372 and 373
  • Epitope B is at positions 333 and 382
  • Epitope C is at positions 340 and 376
  • Epitope D is at positions 391, 392, 393, 394, 395, 396 and 397
  • Epitope E is at positions 355, 356, 357, 359, 364, 365, 404, 407, 412 and 413.
  • mice were immunized with chimeric GII.4 V Ps and the sera were used to test the immune response.
  • GII.4-2006.87A contains the A epitope from 1987 in the 2006 background.
  • GII.4- 2006.87A.02E contains the A epitope from 1987 and the E epitope from 2002 in the 2006 background, as shown in FIG 2.
  • mice were immunized with 10 ul (1 x 10 4 ) VRPs expressing different parental GII.4 capsids or chimeric GII.4 capsids (FIG. 3). Mice were immunized with VRPs and boosted with the same VRPs on day 21. Seven days post-boost, mice were euthanized and serum was collected to use in ELIS A-based assays to determine serum blockade response and reactivity to various VLPs (FIGS. 4A-C).
  • FIGS, 4A-C show sigmoidal curves of the blockade data.
  • the dotted line indicates 50% blockade, the point at which the sera are considered blockade sera. Lines that do not cross the 50% mark indicate the inability of the sera to block that particular VLP. The further left the curve is shifted, the more efficiently the sera blocked that particular VLP.
  • chimeric VLP GII.4-2006.87A, 2006.87A.02E
  • a trivalent mix of VLPs GII.4-1987+2002+2006
  • mice immunized with a single parental VLP (GII.4-1987, 2002, 2006, or 2009) were not able to block all of the VLPs tested (FIGS. SA-D).
  • FIGS. 6A-C represent the concentration of serum at which each sample reaches a blockade response (crosses the dotted line in the curve graphs— this is the EC50 or 50% blockade). Dots below the line indicate a blockade response for that VLP. The lower the point is on the graph, the better the blockade response of the serum. Sera from mice immunized with either chimera (GIL4-2006.87A, GII.4-2006.87A.02E) or the multivalent 1987+2002+2006 cocktail (GII.4-87/02/06) were able to block all VLPs tested, although to varying degrees.
  • mice immunized with a single parental strain VLP could not induce a blockade response against all tested strains (FIGS. 7A- D). Dots above the line indicate a VLP that was not blocked even at the highest serum concentration tested (5%).
  • Table 3 depicts a summary of blockade responses of parental, chimeric and multivalent sera. These data show that monovalent VLP vaccines designed after strains circulating between 1987-2009 (GII.4) elicit poor cross protection to related strains.
  • Multivalent vaccines (multiple VLPs, or multiple V Ps encoding different NoV VLP) give broader responses.
  • a VLP has been designed with one or multiple time-ordered GII.4 epitopes that elicited a broadly neutralizing antibody response that captured more unique strains.
  • FIG. 8 The identification of a GII.4 norovirus conserved blockade epitope using human monoclonal antibodies is shown in FIG. 8.
  • Monoclonal antibodies NVB 71 and NVB1 11 were assayed for the ability to block VLP interaction with carbohydrate ligand.
  • Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to PGM in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment). Error bars represent SEM.
  • Bioinformatic approaches identified five putative antibody epitopes on the surface of GII.4 noroviruses that appeared to be evolving over time and to correlate with the emergence of new GII.4 outbreak strains. Predicted epitopes were expanded to include interacting amino acids within 8A. Epitope A, Epitope B, Epitope C, Epitope D, Epitope E and HBGA binding sites mapped onto the P domain dimer of GI1.4.2002.
  • Hu mAb PGM binding blockade activity against GII.4.1987 epitope-exchanged VLPs for NVB 114 (FIG. 9A) and Epitope A exchanged between GII.4.1987 and GII.4.2006 (FIGS. 9C and E).
  • Sigmoidal curves were fit to the mean percent control binding calculated by comparing the amount of VLP bound to PGM in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard error of the mean.
  • Mean EC50 for blockade of each GII.4.1987 exchange epitope (FIG. 9B) and Epitope A exchange VLPs (FIGS. 9D and F). * VLPs with EC 50 values significantly different from the EC50 for each parental VLP.
  • FIG. 10A is a structural model showing the location of previously-established epitope A residues (grey) and newly identified epitope A residues (black).
  • Sigmoidal curves were fit to the mean percent control binding calculated by comparing the amount of VLP bound to PGM in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard error of the mean (FIG. 11 A). Mean EC50 for blockade of each blocked VLP. * indicates VLPs with significantly different EC50 compared GII.4.2006 (FIG. 11B).
  • PD302 has a D391N change compared to both GII.4-2006b and P.D1.
  • GII.4-2006b is shown in FIGS. 12A and D
  • P.D1 is shown in FIGS. 12B and E
  • P.D302 is shown in FIGS. 12C and F.
  • Residues 390-395 are shown in dark grey for GII.4-2006b and in light grey for P.D1 and for P.D302. Dotted lines represent structure-based predicted polar interactions. Dark residues represent positions that interact with HBGAs (FIGS. 12D-F).
  • VLPs engineered to contain variations of Epitope E were assembled to test the impact of the epitope and the non-epitope backbone on anti-GII.4-2002-G6 binding and
  • FIG. 13A Anti-GII.4-2002-G6 EIA reactivity to the Epitope E constructs. * indicates chimeric VLPs with reactivity significantly different from parental VLPs (FIG. 13B). Blockade of Epitope E constructs binding to PGM by anti-GIL4-2002-G6. The mean g/ml mAb needed to block 50% of GO.4 VLP binding to ligand is indicated by the line in the box. The upper and lower whiskers of the box represent the maximum and minimum values. * indicates VLPs with BT 50 significantly different from GII.4-2002 (FIG. 13C).
  • Epitope E was defined by variable residues 407, 412 and 413 (406, 41 1 and 412 for GII.4.1987) and all residues within 8 angstroms of these sites, as these additional residues are likely to be impacted by the structural differences (driven by the variable sites) that contribute to mAb recognition differences.
  • GII.4.1987 E epitope (FIG. 14A) differs from GII.4.2002 (FIG. 14B) at positions 406/407 and 355. The N to S difference at 406/407 likely alters the rotameric position of R41 1 in the GII.4.2002 VLP, allowing it to extend further from the surface.
  • FIG. 14C GII.4.1987 and GII.4.2002 superimposed.
  • GII.4.2002 differs from GII.4.2006 (FIG. 14E) at five positions in the expanded epitope (positions 355-357, 412 and 413). Superimposition suggests that R41 1 of GII.4.2006 is buried, and variation at 355-357 may alter key interactions involving H357 and D355.
  • FIG. 14F GII.4.2006.N412T superimposed upon GII.4.2006.
  • R41 1 Two potentially important residues are R41 1, which is more surface exposed in GII.4-2006.N412T, and T412, which is buried (FIG. 14G). GII.4.2002 superimposed on GII.4-2006.N412T (FIG. 14H). R41 1 is nearly identical suggesting that N412T frees the R41 1 side chain to extend away from the surface where it likely interacts with the mAb. Resides that regulate R41 1 make up site 1 , and 72% of binding can be recovered with modifications to R411.
  • Site 2 is the second site and is comprised of residues 355-357, and particularly D355 which adds negative potential to the second site.
  • Expanded epitopes include residues within 8 angstroms of the confirmed epitope signatures including residues validated in follow up studies (FIG. 15).
  • 16B is comprised of three charged amino acids that are found at positions 316, 484, and 493 in the conserved region that is predicted to interact with NVB 71.4. Variation at position 310 is proximal to the conserved region (FIG. 16B) and may regulate binding to this conserved site.
  • NVB 71.4 (FIGS. 17A and D), GIIA2002.G5 (FIGS. 17B and E) and NVB 71.4 Fab (FIGS. 17C and F) were assayed for ability to block carbohydrate ligand interaction of GII.4.2006 VLPs at room temperature (black circles) and 37 °C (black squares) and
  • Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment) and the mean EC50 ⁇ g/ml) titer for blockade calculated and compared.
  • the fold change in EC50 titer was defined as the mean ECso at 37 °C compared to room temperature.
  • Mean EC50 blockade titer for GII.4.2006.ERK significantly different from the mean EC50 blockade titer for GII.4.2006 at the same temperature.
  • Non-blockade VLPs were assigned an EC50 of 2X the upper limit of detection for statistical analysis and denoted by a data marker on the graph above the dashed line (assay upper limit of detection) for visual comparison. Error bars represent the SEM on sigmoidal fit curves and 95% confidence intervals on Mean EC50 graphs.
  • the ERK motif was mapped onto the crystal structure of GII.4.2004 to identify sites that may be interacting with the ERK motif (FIG, 18 A).
  • the ERK motif is highly conserved among epidemiologically important GII.4 strains while residue 310 has evolved in the most recent GII.4 strains with global distribution (FIG. 18B).
  • GII.4.2002.G5 (FIGS. 19A and B), and NVB 71 A Fab fragments, and NVB 71.4 IgG (FIG. 19B) were assayed for ability to block the interaction of VLPs with carbohydrate ligand at room temperature.
  • Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment) and the mean EC50 ⁇ g/ml) titer for blockade calculated.
  • the fold change in potency (EC50 titer) was defined as the ratio between mutant VLPs and wildtype VLP at room temperature (FIGS. 19A and B).
  • the fold change in temperature sensitivity was defined as the change in ratio between mean EC 50 at 37 °C compared to room temperature for the mutant VLP compared to ratio at both temperatures for the wildtype VLP (FIG. 19A).
  • FIG. 2 A Sequences from GII.4-1987, GII.4-2002, and GIL4-2006 were aligned and residues in blockade epitopes A, D, and E are shown in FIG. 2 A.
  • Chimeric VLP P2 dimers are shown in FIG. 2C.
  • Differential Epitope A residues from parental strain GII.4-1987 (A294V, R297H, N298D, S368T, E372N) are inserted into the parental GII.4-2006 strain background to create chimeric VLP GII.4-2006.87A.
  • Chimeric VLP GII.4-2006.87A.02E contains the same GII.4-1987 residue substitutions as well as Epitope E substitutions (N412T, V413G).
  • FIG.20 EC50 blockade titers from mice immunized against different GH.4 VLPs are shown in FIG.20.
  • Sera from mice immunized with chimeric VLPs GIL4-2006.87A and GII.4-2006.87A.02E block all VLPs tested from 1987- 2012 (FIG. 20B).
  • FIG. 20C A schematic showing the chimeric VLP vaccine strategy utilizing blockade epitopes from multiple circulating GII.4 strains is shown in FIG. 20C.
  • FIGS. 21- 22 A strategy for the design and reformulation of chimeric VLPs is shown in FIGS. 21- 22.
  • epidemiological studies identify sequence changes in epitope A in minor variants that arise in the population.
  • a panel of VLPs representing this genetic diversity is built and monoclonal antibodies and polyclonal sera can be used to evaluate changes in antigenicity for each variant.
  • specific variants that are antigenically distinct from the predominant strain are identified and can be more closely monitored in the population. (4) If one of these variants seems likely to emerge as a new predominant strain, the chimeric VLP is already made and can quickly be introduced into a new vaccine formulation.
  • VLPs virus replicon particles
  • VLPs were identified from stool samples from multiple infected individuals. Further, all VLPs bind to human mabs and saliva, synthetic HBGAs and pig gastric mucin.
  • GII.4.2002.G5 is a mouse mab generated by hyperimmunization with GII.4.2002 VLPs, as described (41). This antibody is now commercially available from Maine Biotech (MAB227P). Fabs were obtained by papain cleavage using papain immobilized on beaded agarose resin (30 IU/mg) (Pierce) followed by HiTrap protein-A (GE Healthcare) and size-exclusion chromatography
  • Blocking Of Binding (BOB) Assay For experiments using human polyclonal serum, human mabs were purified on protein A or G columns (GE Healthcare) and biotinylated using the EZ-lmk NHS-PEO solid-phase biotinylation kit (Pierce). The competition between polyclonal serum antibodies and biotinylated humabs for binding to immobilized VLPs (1 g/ml) was measured by EIA. Briefly, plasma samples were added to GII.4.1997 or
  • biotinylated humab was added at a concentration corresponding to 80% of the maximal OD level, and the mixture was incubated at room temperature for 1 hour. Plates were then washed with PBS-0.05% Tween- 20 and bound biotinylated humab was detected using AP-labeled streptavidin (Jackson Immunoresearch). The percentage of inhibition was tested in duplicates and calculated as follows: (H(OD sample-OD neg ctr) / (OD pos ctr-OD neg ctr)))xl00.
  • BD 80 value was calculated by interpolation of curves fitted with a 4-parameter nonlinear regression.
  • the binding titers to respective coated VLPs were determined by EIA by measuring the dilution required to achieve 50% maximal binding (EC 50 ) as previously described (52). EIA plates were coated at 0.25 ⁇ g ml VLP for human mab BOB of mouse mab assays.
  • Gil strains and GII.4 strains (28) from 1974 to 2012 were aligned using ClustalX version 2 (75) and the amino acid residues that were conserved among all GO capsid sequences and all GII.4 capsid sequences were mapped onto the GII.4.2004 (PDB accession: 3JSP) (65) crystal structure to identify areas that were conserved among all Gil and all GII.4 capsid proteins.
  • the original analysis was performed using the crystal structure for GII.4.1997 as the distances in the structure used for making the epitope prediction would be more reliable than in a homology model.
  • the ERK and EHNQ motifs were identified as regions that were conserved among Gil noro viruses, and highly conserved among GII.4 viruses.
  • E316, R484, and K493 (ERK) and E488, H501, N522 and Q523 (EHNQ) were identified as conserved residues in these regions that carried a charge and had exposed side chains that protruded. These sites were targeted for mutagenesis using the rationale that preserving the charge of these residues would preserve the structural components necessary for VLP formation.
  • VLP-Carbohydrate Ligand-Binding Assay VLP-Carbohydrate Ligand-Binding Assay.
  • EIA plates were coated with 10 ⁇ Pig Gastric Mucin (PGM) for 4 hours and blocked over night at 4°C in 5% dry milk in PBS-0.05% Tween-20 before the addition of increasing concentrations of VLP.
  • Bound VLP were detected by a rabbit anti-GII.4 norovirus polyclonal sera made from hyperimmunization with a cocktail of GII.4.1987, GII.4.2002, GII.4.2006 and Gil .4.2009 VLPs, followed by anti-rabbit IgG-HRP (GE Healthcare) and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher).
  • VLP-Carbo hydrate Ligand-Binding Antibody Blockade Assay VLP-Carbo hydrate Ligand-Binding Antibody Blockade Assay.
  • VLPs (0.25 ⁇ / ⁇ ) were pretreated with decreasing concentrations of test mab for 1 hour before being added to the carbohydrate ligand-coated plates for 1 hour. Wash steps and bound VLP were detected as described above. The percent control binding was defined as the binding level in the presence of antibody pretreatment compared to the binding level in the absence of antibody pretreatment multiplied by 100.
  • Antibody- VLP and VLP -PGM incubations were done at room temperature or 37°C, as described for each figure. All other incubations were done at room temperature. Antibodies were tested for blockade potential against GIL VLPs at two-fold serial dilutions.
  • EC 50 values were calculated for antibodies that demonstrated blockade of at least 50% at the dilution series tested. Monoclonal antibodies that did not block 50% of binding at the highest dilution tested were assigned an EC50 of 2X the assay upper limit of detection for statistical comparison. EC 50 values between VLPs were compared using the One-way ANOVA with Dunnett posttest, when at least three values were compared or a student's T test when only two values were compared. A difference was considered significant if the P value was ⁇ 0.05.
  • VLP concentrations in blockade assays are in the low nanomolar range and therefore cannot discriminate between antibodies with sub-nanomolar affinities.
  • Antibody- VLP interactions were validated for compliance with the law of mass action by preforming blockade assays of GII.4.1997 and GII.4.2006 at 0.25, 0.5, 1 and 2 ⁇ VLP.
  • EC 50 values for antibody blockade varied less than 2-fold (1 dilution) between any combination of VLP concentration tested, indicating that under the test conditions, antibody is in excess to the VLP and the tenants of the law of mass action are met for the antibody- VLP binding.
  • Blockade assays using human type A or B saliva as the source of carbohydrate ligand were performed as described (76) with 0.5 ⁇ g/ml VLP at room temperature and 37°C.
  • Antibody relative affinity measurements Antibody 3 ⁇ 4 measurements were done as previously described (67) at room temperature and 37°C. Briefly, EIA plates were coated with 0.25 ⁇ g/ml VLP in PBS, blocked, and incubated with serial dilutions of test antibody. Bound antibody was detected by anti-human IgG-HRP and color developed as described above. 3 ⁇ 4 values were calculated using one-site specific binding equation in GraphPad Prism 6. a values were validated by repeating the above assay at a range of VLP concentrations. There was less than a two-fold difference in calculated K d for any concentration between 0.13 and 1.0 ⁇ g/ ⁇ ml for any antibody tested confirming that the antibody was in excess of the VLP under test conditions.
  • VLP-Protein A gold staining VLPs were incubated with 5 g/ml human mab followed by 1/100 dilution of Protein A conjugated to 10 nm gold particles, absorbed onto prepared grids, stained with 2% Uranyl acetate and visualized by TEM. Staining specificity was validated by counting fifty fields of the negative control (VLP minus human IgG plus Protein A-gold). Only one gold particle was observed near a VLP in the fifty negative control fields.
  • Antibodies to conserved NoV epitopes are rarely detected in human serum samples. Monoclonal Abs and polyclonal sera raised in response to mouse immunization or human infection have identified epitope A as the immuno-dominant component of the GII.4 antibody blockade response, accounting for 40-50% of total blockade activity in serum (47, 59). To estimate the fraction of antibodies specific for conserved GII.4 epitopes in the overall serum antibody response, one hundred serum samples collected from healthy individuals were assayed for ability to block binding of human mabs NVB 61.3 and NVB 71.4 in a blockade of binding (BOB) assay (60, 61).
  • BOB blockade of binding
  • the conserved GH.4 blockade epitope is likely not surface exposed and antibody access to the epitope is regulated by particle conformation.
  • NVB 71.4 To characterize the epitope recognized by NVB 71.4 we began by comparing the profile for NVB 71.4 blockade of a time-ordered panel of GII.4 VLPs representing circulating GII.4 strains from 1987 through 2012. As shown previously (52), blockade curves had relatively shallow slopes (range 0.68- 0.92) ( Figure 24A). These data suggest that access of NVB 71.4 to the conserved blockade epitope may be restricted under the test conditions.
  • Viruses and virus-like particles are dynamic structures and the degree of structural flexibility is temperature sensitive in at least some cases (62, 63). Moreover, studies with flaviviruses have demonstrated that the source of virus particles (e.g., cell lines) and host expression patterns (e.g., furin proteases) may also alter particle conformation or dynamics (64). While this study is the first to show that VLPs produced from VEE replicons adopt different conformations, to our knowledge no studies have demonstrated that viruses or VLPs assembled in the baculovirus insect cell system which functions at 27-28°C, are similarly dynamic.
  • source of virus particles e.g., cell lines
  • host expression patterns e.g., furin proteases
  • GII.4.2009 capsid constructs is identical (GenBank accession number ADD10375) in both constructs.
  • blockade of surface epitope A was efficient and not temperature sensitive ( ⁇ 1.3-fold less antibody needed for 50% blockade) ( Figure 26A).
  • NVB 71.4 blockade was also not temperature sensitive (1.3-fold more antibody at 37°C) for the insect cell-produced VLPs, compared to 6.9-fold less antibody needed at 37°C for the mammalian cell produced VLP, Further, NVB 71.4 blockade of GII.4.2009 VLP produced in insect cells required 29.3-fold less antibody for 50% blockade at room temperature and 3.2-fold less at 37°C compared to GII.4.2009 VLPs produced in mammalian cells (0.1 133 and 0.1503 ⁇ compared to 3.322 at room temperature and 0.4817 ⁇ g/ml at 37°C ( Figures 26B and 24 and (52)).
  • GII.4.2002.G5 needed 93-fold less antibody at room temperature and 19-fold less at 37°C for 50% blockade for GII.4.2009 VLPs produced in insect cells compared to mammalian cells (0.0843 and 0.1173 ⁇ g ml compared to 7.8 ⁇ g/ml at room temperature and 2.177 ⁇ / ⁇ at 37°C ( Figures 26C and 25). This lack of temperature effect on Bac-GII.4.2009 blockade was maintained when B saliva was used as the Hgand source and when NVB 71.4 Fab fragments were used for the blocking antibody.
  • This region contained several conserved amino acids in an area large enough to represent a potential antibody binding site (>1000 2 A), including charged amino acids at positions E316, R484, and K.493 (post- 1997 GII.4 numbering) (Figure 16C). These amino acids were named the ERK motif ( Figure 16C).
  • the ERK motif is highly conserved among GII.4 strains that circulated between 1987 and 2012 and was predicted to be either a binding site for or a regulator of NVB 71.4 binding.
  • amino acid position 310 was identified as a site of variation among contemporary GII.4 epidemic strains (2009 and 2012) that was proximal to the highly conserved region containing the ERK motif (Figure 16D).
  • GII.4.2006.EHNQ contains mutated residues E488D, H501K and N522Q and Q523N (post-1997 GII.4 numbering) ( Figure 27A).
  • the GII,4.2006,ERK substitutions did not notably alter particle structure as measured by electron microscopy visualization and ligand binding ability;
  • the GII.4 conserved ERK motif impacts NVB 71.4 and GH.4.2002.G5 blockade capacity with little impact on temperature sensitivity.
  • ERK substitutions resulted in minimal increases in blockade ability for both epitope A and D antibodies (1.3-fold less antibody needed for 50% blockade at 37°C compared to room temperature for both human mabs, data not shown).
  • the ERK motif substitutions resulted in complete loss of blockade potency of NVB 71.4 at room temperature.
  • GII.4.2006 even at the elevated temperature (3.6-fold more). Further, blockade of GII.4.2006 and GII.4.2006.ERK with NVB 71.4 Fab fragments was more potent (lower EC 50 value) but similarly temperature sensitive compared to NVB 71.4 IgG.
  • the EC50 values were 2.1-fold different at room temperature (1.758 verses 0.8052) and 1.4-fold different at 37°C (0.1807 compared to 0.1259), indicating that with the smaller epitope-binding molecule, the ERK residues do not effect antigenicity ( Figure 17C), Further, ERK substitutions negatively impact blockade potency for both conserved epitope antibodies but do not negate the compensatory effect of incubating at higher temperature, indicating that the ERK residues may be affecting antibody access to the epitope instead of the antibody binding strength for the epitope.
  • Quantitative EIAs (67) further indicate that ERK residue substitutions do not affect antibody affinities. Based on the differences in blockade titer, if the ERK substitutions were primarily affecting antibody affinity we would expect a 10-fold change in functional affinity for NVB 71.4 at room temperature and a 4-fold change at 37°C. However, there is less than a two-fold difference (one serial dilution) between antibody functional affinities (3 ⁇ 4 values) of NVB 71.4, GII.4.2002.G5 and epitope D human mab for GII.4.2006 and GII.4.2006.ERK VLPs between room temperature and 37°C (Table 4).
  • Residue 310 modulates antibody blockade potency and temperature sensitivity.
  • GII.4.2002.G5 GII.4.2002.G5, respectively. Conversely, GII.4.2012.D310S blockade potency increased 2.7 and 3.2-fold and temperature sensitivity decreased 4.6 and 3.1 -fold for each antibody. NVB 71.4 Fab had modestly increased potency at room temperature (1-2.8 fold) for the 310 mutant VLPs and the blockade was less temperature sensitive (2.4-8.0 fold) compared to wildtype, indicating that the smaller molecule has better access to the epitope.
  • GII.4.2009.ERK containing both the S310D and ERK substitutions (S310D, E316D, R484K and K493R) ( Figure 28 A).
  • This VLP is called ERK, instead of SERK because of the asparagine found at 310 in the GII.4 VLPs from 1987-2006.
  • combining the 310 and ERK residue changes in the GII.4.2009 backbone resulted in a VLP that was similarly blocked as GII.4.2009 for NVB 71.4 but required 4.1 -fold more GO.4.2002.G5 for 50% blockade.
  • Blockade by anti-epitope A and D human mabs was unaffected by the 310 or ERK residue mutations indicating that the substitutions were specifically targeting the conserved blockade epitopes and not causing global particle disturbances.
  • NVB 71.4 VLP-ligand interaction blockade is not explained by particle disassembly or steric hindrance. The mechanism of antibody blockade of VLP binding to carbohydrate ligand is unknown.
  • NVB 71.4 blockade we stained GII.4.2009 and GII.4.2009.ERK VLPs with NVB 71.4 and epitope A human mabs and Protein A gold particles and observed antibody labeled VLPs by negative stain electron microscopy ( Figure 29). Both NVB 71.4 and the epitope A human mabs labeled intact VLPs, indicating that the antibody-induced lack of ligand binding was not the result of antibody-mediated particle disassembly or that NVB 71.4 preferentially binds to
  • Binding of the epitope A human mab did not affect binding of the mouse mab GII.4.2002.G5 for either VLP.
  • a strain mismatched epitope A human mab did not affect binding of either the mouse epitope A or GII.4.2002.G5 antibody binding for either VLP.
  • pre-incubation of the VLP with NVB 71.4 did not affect binding of the mouse epitope A mabs but decreased binding of Gil A2002.G5.
  • NVB 71.4 human mab blocked 50% of binding of mouse GII.4.2002.G5 at 0.0982 ⁇ for GII.4.1997 and 0.1913 ⁇ for GII.4.2006.
  • FIG 31 shows the mapped epitopes and NERK motif of GII.4 noroviruses.
  • the previously described evolving antibody blockade epitopes A-E are shown on the surface next to the carbohydrate binding sites (circled) (Panel A).
  • the NERK motif is distal to the carbohydrate binding sites (denoted by arrows) (Panel B).
  • FIG. 32 shows a proposed model for regulation of antibody access to the conserved
  • GII.4 blockade epitope(s) by the ERK motif and VLP structural conformation.

Abstract

The present invention provides methods and compositions comprising a chimeric norovirus capsid protein comprising a norovirus VP1 major capsid protein backbone comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the norovirus epitope and/or motif is from a norovirus strain that is different from the norovirus VP1 major capsid protein backbone.

Description

METHODS AND COMPOSITIONS FOR NORO VIRUS BLOCKADE EPITOPES
STATEMENT OF PRIORITY
This application claims the benefit, under 35 U.S.C. §1 19(e), of U.S. Provisional Application No. 61/798,946, filed March 15, 2013 and U.S. Provisional Application
No.61/887,101, filed October 4, 2013, the entire contents of each of which are incorporated by reference herein.
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under Grant No. AI 056351 awarded by the National Institutes of Health. The United States government has certain rights in the invention.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
A Sequence Listing in ASCII text format, submitted under 37 C.F. . § 1.821, entitled 5470-659TS_ST25.txt, 61,251 bytes in size, generated on March 13, 2014 and filed via EFS- Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
FIELD OF THE INVENTION
The present invention relates to compositions comprising norovirus blockade epitopes and methods of their use in therapeutic and diagnostic applications.
BACKGROUND OF THE INVENTION
Noroviruses are the leading cause of severe viral gastroenteritis and are responsible for 50% of all acute gastroenteritis outbreaks in the United States and Europe [1], Although the severity of disease is usually moderate, lasting 1-3 days, infection can be especially virulent in young children, the elderly, and the immunocompromised, with the latter group experiencing chronic diarrhea and vims shedding for over a year [2-8], Importantly, it is estimated that 200,000 people die each year from norovirus infections, primarily children in the developing world [9]. An effective vaccine would be particularly advantageous for the very young and aged populations, military personnel, children and healthcare providers, food handlers, cruise ship passengers, and populations of the developing world [10],
Immunotherapeutics are especially needed for treating immuno suppressed populations experiencing long-term infections with chronic diarrhea. The lack of understanding of the extensive antigenic relationships among the large number of norovirus strains and the complex relationship between host protective immunity and virus antigenic heterogeneity are the primary obstacles to norovirus vaccine development.
The present invention overcomes previous shortcoming in the art by providing norovirus blockade epitopes and methods of their use in therapeutic and diagnostic applications.
SUMMARY OF THE INVENTION
The present invention provides a chimeric norovirus capsid protein comprising a norovirus VP1 major capsid protein backbone comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the norovirus epitope and/or motif is from a norovirus strain that is different from the norovirus VP 1 major capsid protein backbone.
Also provided herein is a synthetic backbone molecule comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the backbone molecule allows for formation of a conformational epitope and/or motif.
Further provided herein is a norovirus P particle comprising a norovirus epitope and/or selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the epitope and/or motif is presented on the P particle surface.
In addition, the present invention provides a mimitope comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above.
The present invention further provides therapeutic methods. Thus, in one
embodiment, the present invention provides a method of producing an immune response to a norovirus in a subject, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention. Also provided herein is a method of treating a norovirus infection in a subject in need thereof, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention.
Further provided herein is a method of preventing a disease or disorder caused by a norovirus infection in a subject, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention.
In additional embodiments, the present invention provides a method of protecting a subject from the effects of norovirus infection, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention, the VLP of this invention and/or the composition of this invention.
The present invention also provides diagnostic methods. Thus, in one aspect, the present invention provides a method of detecting a neutralizing antibody to a norovirus, the method comprising determining whether an antibody binds to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention, wherein binding by the antibody to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention indicates that the antibody is a neutralizing antibody to a norovirus.
A method is also provided herein of identifying a neutralizing antibody to a norovirus, comprising: (a) contacting an antibody with the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention; and (b) determining if the antibody binds to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention, wherein binding by the antibody to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention identifies the antibody as a neutralizing antibody to a norovirus.
In further embodiments, the present invention provides a method of identifying an immunogenic composition that induces a neutralizing antibody to a norovirus in a subject, the method comprising: (a) contacting a biological sample from a subject that has been administered the immunogenic composition with the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention;
(b) determining if the biological sample comprises an antibody that binds the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention; and (c) identifying the immunogenic composition as inducing a neutralizing antibody to a norovirus in the subject if the biological sample comprises an antibody that binds to the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention.
Also provided herein is a method of identifying an immunogenic composition that induces a neutralizing antibody to a norovirus in a subject, the method comprising: (a) administering an immunogenic composition comprising a norovirus antigen to a subject in an amount effective to induce antibodies against the norovirus antigen; (b) contacting a biological sample from the subject with the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention;
(c) determining if the biological sample comprises an antibody that binds the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention; and (d) identifying the immunogenic composition as inducing a neutralizing antibody to a norovirus in the subject if the biological sample comprises an antibody that binds the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the VRP of this invention and/or the VLP of this invention. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the evolutionary profile of epitopes of various human norovirus (NoV) strains. The evolution of the GII.4 noro viruses was assessed over a 36 year period of time by comparing strains from 1 74 to 2010. In comparing these sequences, sites of variation in the p2 subdomain were noted, and these sites were mapped onto the crystal structure of the P- domain dirner for the 1997 strain VA387, Surface-exposed sites of variation were then examined to determine which residues were close enough to constitute a single epitope, and epitopes A-E and the NERK motif (referred to previously as Epitope F, this motif has been shown to influence binding of broadly neutralizing antibodies such as NVB 71.4 and
GII.4.2002.G5 and has been identified to be a combination of amino acid residues that regulate antibody binding capability) were identified based upon this variation. VLPs containing substituted Epitopes A through E and the NERK motif were created and tested for ability to bind to surrogate neutralizing antibodies in the VLP-HBGA blockade assay.
Epitopes A, D and E and the NERK motif were confirmed to be blockade epitopes.
Numbering of amino acids is based on amino acid position of the full amino acid sequence of the norovirus strains listed as provided in the GenBank® Database or as provided herein. For example, the numbering of amino acids can be based on the amino acid sequence of
GII.4.2002 (GenBank® Accession No. AFJ04708.1 for amino acid sequence; GenBank® Accession No. JQ478408 for nucleotide sequence), provided herein as SEQ ID NO:4. For Gl 1.4.1974, GII.4.1987 and GII.4.1997, the amino acids of Epitope E are 406, 41 1 and 412. The amino acids for Epitope E of the other listed norovirus strains are 407, 412, and 413 as shown, based on an insertion of an amino acid at position 394 in these subsequently listed strains. For the same reason, the amino acid residues for Epitope D will be at alternative corresponding positions that would be readily recognized by one of ordinary skill in the art.
FIG. 2 depicts chimeric Venezuelan equine encephalitis replicon particles (VRPs) used to immunize mice.
FIG. 3 depicts the immunization of mice with GII.4 parental or chimeric capsids. FIG. 4 depicts blockade data with: (A) anti-2006.87A mouse sera; (B) anti- 2006.87A.02E mouse polyclonal sera; and (C) anti-2006-87A.02E mouse polyclonal sera on various VLPs.
FIG. 5 depicts blockade data with: (A) anti-1987 mouse sera; (B) anti-2002 mouse sera; (C) anti-2006 mouse sera; and (D) anti-2009 mouse sera on various VLPs. FIG. 6 depicts the EC50 at which each sample reaches a blockade response for: (A) anti-GII .4-2006.87A mouse sera; (B) anti-GIL4-2006.87A.02E mouse sera; and (C) anti- Gil .4-2006-87/02/06 mouse sera with various virus like particles (VLPs).
FIG. 7 depicts the EC50 at which each sample reaches a blockade response for: (A) anti-GII.4-1987 mouse sera; (B) anti-Gil 4-2002 mouse sera; (C) anti-GII.4-2006 mouse sera; and (D) anti-GII.4-2009 mouse sera with various VLPs.
FIG. 8 depicts the results of initial studies to identify a GII.4 norovirus conserved blockade epitope using human monoclonal antibodies.
FIG. 9 depicts that Epitope A comprises an evolving GIL4 blockade epitope recognized by NVB 114, 11 1 and 43.9,
FIG. 10 shows that residues 292, 295, and 373 are part of Epitope A.
FIG. 11 shows that amino acids 393-395 comprise a blockade epitope for
contemporary GIL4 strains recognized by NVB 97.
FIG. 12. Depicts that changes in amino acid position 391 alter Epitope D polar interactions, identifying 391 as a likely part of Epitope D
FIG. 13 depicts the characterization of Epitope E.
FIG. 14. depicts that the variation in Epitope E alters the structure of the caps id, identifying residues 355 and 357 as a likely part of Epitope E.
FIG. 15 depicts the expanded GII.4 blockade antibody epitopes over time.
FIG. 16 depicts the structural model of a conserved epitope.
FIG. 17 shows that ER motif substitutions decrease NVB 71.4 and GII.4.2002.G5 blockade potential with little impact on blockade temperature sensitivity.
FIG. 18 depicts the identification of residues that interact with the ERK motif.
FIG. 19 shows that residue 310 inversely modulates blockade potency and temperature sensitivity of the conserved GIL4 epitope.
FIG. 20 shows that chimeric VLPs induce a broadened blockade response.
FIG. 21 depicts a model of chimeric GII.4 VLP-based vaccine design and
reformulation strategy.
FIG. 22 depicts a method for the rational design of broadly cross-protective vaccines. FIG 23. Antibodies to conserved NoV epitopes are rare in human plasma. The ability of human serum samples (n=100) to block binding of human monoclonal antibodies (mabs) was evaluated using a Blockade of Binding (BOB) assay. Shown is the reciprocal plasma dilution that blocks 80% binding (BDg0) of a conserved GII non-blockade epitope antibody (NVB 61.3) and a conserved GII.4 blockade epitope antibody (NVB 71.4) to
GII.4.1997 (Panel A) and GII.4.2006 (Panel B) VLPs. Each symbol represents a different individual. BDg0 values <40 were scored as negative. Total serum IgG binding to GII.4.1997 and GII.4.2006 was determined by enzyme immunoassay (EIA). Reciprocal EC50 values are shown (right column Panel A and B). · Sera competing for binding of NVB 61.3; · Sera competing for binding of NVB 71.4; * Donor source of NVB 61.3 and 71.4.
FIG 24. Access of NVB 71.4 to the conserved GII.4 blockade epitope is temperature dependent. NVB 71.4 was assayed for ability to block the interaction of a panel of time-ordered GII.4 VLPs with carbohydrate ligand. Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment) o
at room temperature (Panel A) and 37 C (Panel B) and the mean EC50 ^g/ml) titers for o
blockade at room temperature (·) and 37 C (·) calculated and compared (Panel C). The
o
fold change in EC-Q titer was defined as the mean EC-0 at 37 C compared to room
temperature. * Mean EC50 blockade titer is significantly different between room temperature
0
and 37 C. Dashed line in Panel C marks the assay upper limit of detection. Error bars represent the SEM on sigmoidal fit curves and 95% confidence intervals on Mean EC50 graphs.
FIG 25. Access of GII.4.2002.G5 to a conserved GII.4 blockade epitope is regulated by temperature. GII.4.2002.G5 was assayed for ability to block the interaction of
GII.4 VLPs with carbohydrate ligand at room temperature (·) and 37 C (·). Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment) and the mean EC5Q (μ§/τηΥ) titer for blockade calculated. The fold change in EC50 titer was defined as the mean ECS0 at 37 C compared to room temperature.
o
* Mean ECSQ blockade titer is significantly different between room temperature and 37 C, Non-blockade VLPs were assigned an EC50 of 2X the upper limit of detection for statistical analysis and denoted by a data marker on the graph above the dashed line (assay upper limit of detection) for visual comparison. Error bars represent 95% confidence intervals.
FIG 26. Antibody access to the conserved epitope is not temperature sensitive on
GII.4.2009 VLPs made at lower temperature in insect cells. Epitope A human mab (Panel A), NVB 71.4 (Panel B) and GII.4.2002.G5 (Panel C) were assayed for ability to block the interaction of GII.4.2009 VLPs produced in insect cells using a baculovirus expression system and carbohydrate ligand at room temperature (·) and 37 C (·). Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatrnent compared to the amount of VLP bound in the absence of antibody pretreatment) and the mean EC50 (μ /πύ) titer for blockade calculated. The fold change in
EC50 titer was defined as the mean EC50 at 37 C compared to room temperature. * Mean
o
EC50 blockade titer is significantly different between room temperature and 37 C. Error bars represent 95% confidence intervals.
FIG 27. Characterization of VLPs with substitutions in predicted conserved antibody epitopes. Schematic of constructs (Panel A). Particle integrity was verified by transmission electron microscope visualization (Panel B) and carbohydrate ligand (Pig Gastric Mucin type III, PGM) binding of VLPs (Panel C). Non-PGM binding VLPs were assigned an EC50 of 2X the upper limit of detection for statistical analysis and denoted by a data marker on the graph above the dashed line (assay upper limit of detection) for visual comparison. Error bars represent 95% confidence intervals.
FIG. 28. Characterization of VLPs with substitutions in residue 310 and ERK.
Schematic of constructs (Panel A). Particle integrity was verified by transmission electron microscope visualization (Panel B) and carbohydrate ligand (PGM) binding of VLPs (Panel C). Dashed line marks the upper limit of detection in Panel C. Error bars represent 95% confidence intervals.
FIG. 29. Antibody-bound VLPs retain structural integrity. GO.4.2009 (Panel A) and GII.4.2009.ERK (Panel B) VLPs were immuno-stained with NVB 71.4 or epitope A (GII.4.2009 only, Panel C) human mabs and visualized by negative stain transmission electron microscopy. Arrows denote immuno-gold labeled VLPs.
FIG 30. Binding of NVB 71.4 does not disrupt surface epitope A topolo y.
Human mabs to surface epitope A or NVB 71.4 were evaluated for ability to block binding of mouse mabs to epitope A or the conserved blockade epitope in GII.4.1997 (Panel A) and GII.4.2006 (Panel B) using a BOB assay. Sigmoidal curves were fit to the mean percent control binding (percent of mouse mab bound to VLP in the presence of human mab pretreatment compared to the amount of mouse mab bound in the absence of human mab pretreatment) and the mean EC50 fag/ml) titer for blockade of binding calculated, R EC-0 > 8 g ml, " EC50 < 1 μ /ηι1.
FIG 31. The mapped epitopes of GII.4 noroviruses. The previously described evolving antibody blockade epitopes A-E are shown on the surface next to the carbohydrate binding sites (circled) (Panel A). The ERK motif is distal to the carbohydrate binding sites (denoted by arrows) (Panel B).
FIG. 32. Proposed model for regulation of antibody access to the conserved GII.4 blockade epitope/s by the ERK motif and VLP structural conformation.
DETAILED DESCRIPTION OF THE INVENTION
Noroviruses are single-stranded, positive sense RNA viruses that belong to the family Caliciviridae. The norovirus genome is ~7.5 kb and contains three open reading frames. ORF1 encodes the non-structural proteins, ORF2 encodes the VPl major capsid protein, and ORF3 encodes the VP2 minor capsid protein. VPl is divided into three parts: the shell, which forms the irrner-most portion of the virion; the PI subdomain, which forms a staik-like projection away from the virion surface; and the P2 subdomain, which is a highly variable, surface exposed region that sits on top of PI . The P2 subdomain of the VPl major capsid protein contains potential neutralizing antibody epitopes. These epitopes change over time, likely allowing new strains to escape human herd immunity.
An effective GII.4 norovirus vaccine needs to address these antigenic changes over time. The present invention is based on the identification and characterization of various neutralizing blockade epitopes in norovirus capsid protein, which has led, in some embodiments, to the development of a vaccine platform to allow a single immunogenic protein to protect against multiple strains of norovirus. Thus, in one aspect, the present invention provides a chimeric norovirus capsid protein comprising a norovirus VPl major capsid protein backbone comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NER motif; and g) any combination of (a) through (f) above, wherein the norovirus epitope and/or motif is from a norovirus strain that is different from the norovirus ORF2 major capsid protein backbone. Nonlimiting examples of Epitope A, Epitope B, Epitope C, Epitope D, Epitope E and the NERK motif are provided for several norovirus strains, as shown for example, in Figure 1 and Figure 15 herein. Also provided herein is a synthetic backbone molecule comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the backbone molecule allows for formation of the conformational epitope and/or motif.
Further provided herein is a norovirus P particle comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above, wherein the epitope and/or motif is presented on the P particle surface.
In addition, the present invention provides a mimitope comprising a norovirus epitope and/or motif selected from the group consisting of: a) Epitope A; b) Epitope B; c) Epitope C; d) Epitope D; e) Epitope E; f) NERK motif; and g) any combination of (a) through (f) above.
In some embodiments of the chimeric norovirus capsid protein of this invention, the norovirus VP1 major capsid protein backbone can be from any norovirus strain and in some embodiments can be from a genogroup Gil, genotype 4 (GII.4) strain.
In some embodiments, of the chimeric norovirus capsid protein of this invention, the norovirus VP1 major capsid protein backbone can be from GII.4.1974 (GenBank® Database Accession No. ACT76139.1), GII.4.1987 (GenBank® Database Accession No.
AAK50355.1), GII.4.1997 (GenBank® Database Accession No. JQ478407), GII.4.2002a, Gil .4.2002 (GenBank® Database Accession No. JQ478408), Gil .4.2004 (GenBank®
Database Accession No. AAZ31376.2), GII.4.2005 (GenBank® Database Accession No. BAE98194.1), GII.4.2006 (GenBank® Database Accession No. JQ478409), GII.4.2007 (GenBank® Database Accession No. AB496912.1), GII.4.2008s (GenBank® Database Accession No. BAH30707.1), GII.4.2008a (GenBank® Database Accession No.
ACX31885,1), GII.4.2009 (GenBank® Database Accession No. ADD10375), or GII.4.2012 (GenBank® Database Accession No. JX459908).
The amino acid sequence of GII.4.2002a is as follows (SEQ ID NO: 5).
mkmasndanpsdgstanlvpevnnevmalepwgaaiaapvagqqnvidpwirnnfvqapggeftvsprnapgeilwsapl gpdlnpylshlarmyngyaggfevqvilagnaftagkiifaavppnfpteglspsqvtmfphiivdvrqlepvliplpdvmnfyh ynqsndptikliamlytplrarmagedvftvscrvlt^spdfdfiflvpptvesrtksftvpiltveeratnsrfpipk
vqpqngrcttdgvllgttqlspvnictfrgdvthiagthnytnmlasqnwnnydpteeipaplgtpdfvgriqgmltqttrg hkatvstgdvhftpklgsiqfntdtnndfetgqntkf^
ffrstmpgcsgypnmnldcllpqewvqhfyqeaapaqsdvallrfvnpdtgrvlfecklhksgyvtvahtgqhdlvippngyfrf dswvnqfytlaprngngtgrrral In various embodiments, the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope A (amino acids 294, 296, 297, 298, 368 and 372) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012.
In various embodiments, the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope B (amino acids 333 and 382) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GIIA2008a} GII.4.2009 or GII.4.2012.
In various embodiments, the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope C (amino acids 340 and 376) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GO.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GIL4.2008a, GII.4.2009 or GII.4.2012.
In various embodiments, the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope D (amino acids 3 3, 394 and 395) from GII.4.1 74, GII.4.1 87, GII.4.1997, GO ,4.2002a, GII.4.2002, GII.4.2004,
GII.4.2005, GH.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope D is from a norovirus strain different from the norovirus ORF2 major capsid protein backbone.
In various embodiments, the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise Epitope E (amino acids 407, 412 and 413) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope E is from a norovirus strain different from the norovirus ORF2 major capsid protein backbone.
In various embodiments, the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the norovirus P particle of this invention and/or the mimitope of this invention can comprise the NER motif (previously referred to as Epitope F) (amino acids 310, 316, 484 and 493) from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012.
In some embodiments of the chimeric norovirus capsid protein of this invention, the norovirus ORF2 major capsid protein backbone can be from GII.4.2006 and can comprise Epitope A from GII.4.1987 and Epitope E from GII.4.2002.
The present invention further provides an isolated nucleic acid molecule encoding the chimeric norovirus capsid protein of this invention, a vector comprising the nucleic acid molecule of this invention and a cell comprising the capsid protein, nucleic acid molecule and/or vector of this invention.
In some embodiments, the present invention provides a Venezuelan Equine
Encephalitis (VEE) replicon particle (VRP) comprising the nucleic acid molecule of this invention.
In some embodiments, the present invention provides a virus like particle (VLP) comprising the chimeric norovirus capsid protein of this invention.
The present invention also provides a composition comprising the chimeric norovirus capsid protein of this invention, the synthetic backbone molecule of this invention, the P particle of this invention, the mimitope of this invention, the nucleic acid molecule of this invention, the vector of this invention, the VRP of this invention and/or the VLP of this invention in a pharmaceutically acceptable carrier.
In some embodiments of the invention the individual and conformational epitopes of the norovirus capsid proteins can be presented on a synthetic backbone or support structure so that the epitopes within the synthetic backbone or support structure mimic the conformation and arrangement of the epitopes within the structure of the norovirus capsid protein, VLP or VRP.
In still further embodiments of the invention, the present invention provides peptide mimitopes (see, Meloen et al. (2000) J. Mol. Recognit, 13, 352-359) that mimic the individual and conformational epitopes of the norovirus capsid proteins of the invention. Mimitopes may be identified using any technique known in the art, such as by surface stimulation, random peptide libraries or phage display libraries, using an antibody or antibodies to the individual and conformational epitopes of the chimeric norovirus capsid proteins of the invention.
In yet further embodiments of this invention a norovirus P particle {see, Tan et al. (2011) J Virol. 85(2), 753-764) is provided that presents the epitopes of the norovirus capsid proteins as described herein. In an aspect of the chimeric VLP/V P/virus vaccine approach of the invention, one or more of the identified potential neutralization epitopes from one or more donor strains is moved into any other Gil .4 noro virus backbone strain to induce broad protection against multiple strains. In another aspect of the invention, moving epitope A from GII.4.1987 and epitope E from GII.4.2002 into the GII.4.2006 backbone induces a broadly blocking immune response in mice against GII.4 strains GII.4.1987, 2002, 2006, 2009, and 2012. Thus, a vaccine that incorporates epitope(s) from one or more circulating strains into the backbone of another strain can induce protection against multiple norovirus strains.
In a further aspect of the invention, in order to create a chimeric norovirus construct, the full-length ORF2 major capsid gene sequence from norovirus is either cloned from a patient sample or produced as a synthetic construct (e.g., from a commercial source). Natural or engineered endonuclease sites are used to insert sequence containing the desired epitope changes for one or more GII.4 strains. Alternatively, in yet another aspect of the invention, the full-length capsid may be synthesized (e.g., using a consensus sequence) with the desired sequence changes already present. After production of the desired full-length chimeric GII.4 norovirus capsid gene, this gene is then cloned into an expression vector, Upon expression, the VPl major capsid protein self-assembles into VLPs, which can then be purified.
Alternatively, VRPs expressing the major capsid protein can be produced and purified and subsequently used as a vaccine or used as a source of VLP production.
The term "chimeric norovirus capsid protein" and similar terms will be understood in the art to mean a norovirus capsid protein derived from a particular norovirus strain that contains single or multiple amino acid substitutions at various positions in which the amino acid substitution(s) is an amino acid(s) that is one from the corresponding position(s) of a norovirus capsid protein from a different norovirus strain. In representative embodiments, the amino acid substitution comprises a particular epitope from a norovirus strain different from that of the capsid protein in which the substitution is made. In some embodiments, the amino acid substitutions may be at amino acids 292, 294, 295, 296, 297, 298, 357, 365, 368, 372 and 373 (Epitope A) of the norovirus VPl major capsid protein encoded by open reading frame 2 (ORF2) of human norovirus (hNoV), In some embodiments, the substitutions may be at amino acids 333 and 382 (Epitope B) of the hNoV VPl major capsid protein. In some embodiments, the substitutions may be at amino acids 340 and 376 (Epitope C) of the hNoV VPl major capsid protein. In some embodiments, the substitutions may be at amino acids 391, 393, 394, 395, 396 and 397 (Epitope D) of the hNoV VPl major capsid protein. In some embodiments, the substitutions may be at amino acids 355, 356,357, 359, 364, 365, 404, 407, 412 and 413, or the corresponding amino acids (e.g., 406, 41 1 and 412) of certain NoV strains, (Epitope E) of the hNoV VP1 major capsid protein. In still further
embodiments, the amino acid substitutions may be at amino acid positions 310, 316, 484 and 493 (NERK motif, previously referred to as Epitope F) of the hNoV VP1 major capsid protein.
In some embodiments, the amino acid substitutions may comprise any two or more (e.g., 2, 3, 4, or 5) of these epitopes and/or motifs, in any combination in the same norovirus capsid protein. Nonlimiting examples of the epitopes and motifs of this invention that can be combined in the same norovirus capsid protein include the following combinations (wherein A is Epitope A, B is Epitope B, C is Epitope C, D is Epitope D, E is Epitope E and F is the NERK motif): AB, AC, AD, AE, AF, BC, BD, BE, BF, CD, CE, CF, DE, DF, EF, ABC, ABD, ABE, ABF, ACD, ACE, ACF, ADE, ADF, AEF, ABCD, ADEF, ABEF, ACEF, ABCE, ABCF, ABDF, ABCE, ACDF, BCDEF, BCDE, BCD, CDEF, CDE, CDF, DEF, ACDEF, ACDE, ABDEF, ABDE, ABCDEF, ABCDE, ABCDF, ABCD, CDE, ABCEF, ABEF, BCEF, CEF, BEF, BED, BDF and BDE and also include any combination of A, B, C, D, E and F not specifically recited herein. In a particular embodiment, the combination of amino acid substitutions may be for Epitope A and Epitope E (i.e., AE). In some
embodiments, the amino acid residues of a single epitope or motif(e.g., Epitope A, Epitope B, Epitope C, Epitope D, Epitope E, NERK motif) from a GII.4 norovirus strain is substituted into a capsid protein backbone of a different GII.4 norovirus strain.
As explained herein, the present invention is based on the discovery and
characterization of epitopes and motifs in the norovirus GII.4 capsid protein that function as neutralization epitopes (i.e., blockade epitopes) and motifs that induce a protective immune response to GII.4 norovirus strains. The identification of the amino acid residues that make up these epitopes and motifs can be applied in developing a vaccine platform, wherein one or more of these epitopes and/or motifs from one or more donor norovirus strains are introduced into a different GII.4 norovirus capsid protein backbone to produce a chimeric norovirus capsid protein that induces broad protection against multiple norovirus strains. For example, data provided herein demonstrate that moving Epitope A from GII.4.1987 and Epitope E from GII.4.2002 into the GII.4.2006 capsid protein backbone induces a broadly blocking immune response in mice against strains GII.4.1987, GII.4,2002, GII.4.2006, GO.4.2009 and GII.4.2012. Thus, creating a vaccine that incorporates epitopes and/or motifs from one or more circulating strains into the backbone of another strain may induce protection against multiple norovirus strains and/or novel norovirus strains. These blockade epitopes and motifs have been shown to evolve over time in response to herd immunity and thus these epitopes and motifs can be used as a blueprint to update amino acid residues at defined sites that make up these epitopes and motifs in order to provide immunoprotection against newly emerging strains of GII.4 noroviruses. This means that the particular amino acid residue at a site defined on a GH.4 norovirus to be part of or included in an epitope or motif of this invention can be an amino acid residue already known to be present in a GII.4 norovirus strain or it can be any amino acid residue newly identified to be present at a defined site in the capsid protein of an emerging or previously
uncharacterized GII.4 strain. Therefore, the amino acid residue at a given position in the norovirus capsid protein can be any amino acid residue that makes up a blockade epitope or motif. For example, amino acid residue 365 has been identified to be either I or V in the respective GII.4 norovirus strains listed in Figure 15. In order to produce a chimeric norovirus capsid protein of this invention that comprises Epitope A, an I or V substitution can be made in a norovirus capsid protein backbone, in addition to substitutions at the other amino acid residue sites that make up Epitope A, such that the capsid protein backbone comprises all of the amino acid residues that make up Epitope A of a particular GII.4 strain that is different from the backbone strain. However, if an emerging or previously
uncharacterized GII.4 norovirus strain is found to have an amino acid residue at position 365 that is not I or V, such newly identified amino acid residue can be substituted into a norovirus capsid protein backbone at position 365, along with all of the other amino acid residues identified in the emerging or previously uncharacterized norovirus strain at the other amino acid positions that make up Epitope A, in order to produce a chimeric norovirus capsid protein of this invention that can induce an immunoprotective response against not only the emerging or previously uncharacterized GII.4 norovirus strain, but also against other GII.4 norovirus strains. Thus, the present invention provides for the production of chimeric GII.4 capsid proteins as described herein that can be used to immunize subjects against a newly emerging GII.4 strain as soon as the amino acid residues of a blockade epitope or motif of this invention are identified in the new strain.
The term "norovirus capsid protein backbone" and similar terms refer to the particular norovirus capsid protein from which a chimeric norovirus capsid protein is based. The norovirus capsid protein backbone may be from any geno group, genotype and strain of hNoV. In an embodiment of the invention, the norovirus capsid protein backbone is from genogroup II and genotype 4 (GII.4) of hNoV. In an embodiment, the norovirus capsid protein backbone may be from GII.4-1974 (GenBank Access. No. ACT76139.1). In another embodiment, the norovirus capsid protein backbone may be from GO.4-1 87 (GenBank Access. No. AA 50355.1). In another embodiment, the norovirus capsid protein backbone may be from GII.4-1997 (GenBank Access. No. AFJ04707.1 ). In another embodiment, the norovirus capsid protein backbone may be from GO .4-2002 (GenBank Access. No.
AFJ4708.1 ), In another embodiment, the norovirus capsid protein backbone may be from GII.4-2004 (GenBank Access. No. AAZ31376.2). In another embodiment, the norovirus capsid protein backbone may be from GII.4-2005 (GenBank Access. No, BAE98194.1). In another embodiment, the norovirus capsid protein backbone may be from GO.4-2006 (GenBank Access. No. AFJ4709.1). In another embodiment, the norovirus capsid protein backbone may be from GII.4-2007 (GenBank Access. No. BAH56690.1), In another embodiment, the norovirus capsid protein backbone may be from GII.4-2008a (GenBank Access. No. ACX31885.1). In another embodiment, the norovirus capsid protein backbone may be from GO.4-2008s (GenBank Access. No. BAH30307.1). In another embodiment, the norovirus capsid protein backbone may be from GII.4-2009 (GenBank Access. No.
ADD10375.1). In another embodiment, the norovirus capsid protein backbone may be from GIL4-2012 (GenBank Access. No. AFV08795.1).
The epitope of the chimeric norovirus capsid protein may be from any norovirus genogroup, genotype and strain, as long as it different from that of the norovirus capsid protein backbone. In a particular embodiment, wherein the norovirus capsid protein backbone is from GII.4-2006, the epitope is epitope A from GII.4-1987. In another particular embodiment, wherein, the norovirus capsid protein backbone is from GII.4-2006, the epitopes can be a combination of Epitope A from GIL 1987 and Epitope E from GII.4-2002.
The invention further provides a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding a chimeric norovirus capsid protein or a polypeptide of the invention.
The invention further provides a nucleic acid molecule (e.g. , an isolated nucleic acid molecule) encoding a chimeric norovirus VLP, a chimeric norovirus VRP or a viral coat of a chimeric norovirus particle of the invention.
Also provided are vectors encoding the nucleic acid molecules of the invention.
Also provided are cells that comprise the vectors, nucleic acid molecules, norovirus epitopes, polypeptides, chimeric norovirus VLPs, chimeric norovirus VRPs and/or chimeric norovirus particles of the invention.
The invention also provides immunogenic compositions comprising the cells, vectors, nucleic acid molecules, norovirus epitopes, chimeric norovirus capsid proteins, polypeptides, chimeric norovirus VLPs, chimeric norovirus VRPs, and/or chimeric norovirus particles of the invention in a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition is monovalent. In some embodiments, the immunogenic composition is multivalent for different norovirus serotypes.
The invention encompasses methods of producing an immune response to a norovirus in a subject, the method comprising administering to the subject an effective amount of a norovirus epitope, a chimeric norovirus capsid protein, a polypeptide, a chimeric norovirus VLP, chimeric norovirus VR1P or chimeric norovirus particle, nucleic acid molecule, vector, cell and/or immunogenic composition of the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
As used herein, "a," "an" or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a multiplicity of cells.
Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of
combinations when interpreted in the alternative ("or").
As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel character! stic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 21 11.03. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.
The term "about," as used herein when referring to a measurable value such as an amount or concentration (e.g., the amount of a substrate), is meant to encompass variations of ± 10%, ± 5%, ± 1 %, ± 0.5%, or even ± 0.1% of the specified measurable value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measureable value may include any other range and/or individual value therein.
As used herein, the term "nucleic acid" encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g. , chemically synthesized) DNA and chimeras of RNA and DNA. The nucleic acid may be double-stranded or single-stranded. The nucleic acid may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
As used herein, the term "polypeptide" encompasses both peptides and proteins
(including fusion proteins), unless indicated otherwise.
A "fusion protein" is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame.
A "recombinant" nucleic acid, polynucleotide or nucleotide sequence is one produced by genetic engineering techniques.
A "recombinant" polypeptide is produced from a recombinant nucleic acid, polypeptide or nucleotide sequence.
As used herein, an "isolated" polynucleotide (e.g., an "isolated nucleic acid" or an "isolated nucleotide sequence") means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide. Optionally, but not necessarily, the "isolated"
polynucleotide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration). In representative embodiments, the isolated polynucleotide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
An "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide. Optionally, but not necessarily, the "isolated" polypeptide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five -hundred -fold, one thousand-fold, ten thousandfold or greater concentration). In representative embodiments, the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
Furthermore, an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature. For example, an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier.
The terms "immunogen" and "antigen" are used interchangeably herein and mean any compound (including polypeptides) to which a cellular and/or humoral immune response can be directed. In particular embodiments, an immunogen or antigen can induce a protective immune response against the effects of noro irus infection.
"Effective amount" as used herein refers to an amount of a vector, nucleic acid molecule, epitope, polypeptide, cell, composition or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art, As appropriate, an "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
The term "immunogenic amount" or "effective immunizing dose," as used herein, unless otherwise indicated, means an amount or dose sufficient to induce an immune response (which can optionally be a protective response) in the treated subject that is greater than the inherent immunity of non-immunized subjects. An immunogenic amount or effective immunizing dose in any particular context can be routinely determined using methods known in the art.
The terms "vaccine," "vaccination" and "immunization" are well-understood in the art, and are used interchangeably herein. For example, the terms vaccine, vaccination or immunization can be understood to be a process or composition that increases a subject's immune reaction to an immunogen (e.g. , by providing an active immune response), and therefore its ability to resist, overcome and/or recover from infection (i.e., a protective immune response).
By the terms "treat," "treating" or "treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder. In representative embodiments, the term "treat," "treating" or "treatment of (and grammatical variations thereof) refer to a reduction in the severity of viremia and/or a delay in the progression of viremia, with or without other signs of clinical disease.
A "treatment effective" amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
The terms "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject relative to what would occur in the absence of the methods of the invention. In representative embodiments, the terms "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention of viremia in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the methods of the present invention.
A "prevention effective" amount as used herein is an amount that is sufficient to prevent (as defined herein) the disease, disorder and/or clinical symptom in the subject.
Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.
The efficacy of treating and/or preventing a disease or disorder caused by norovirus infection by the methods of the present invention can be determined by detecting a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters (e.g., viremia), as would be well known to one of skill in the art.
Unless indicated otherwise, the terms "protect," "protecting," "protection" and "protective" (and grammatical variations thereof) encompass both methods of preventing and treating norovirus infection in a subject, whether against one or multiple strains, genotypes or genogroups of norovirus.
The terms "protective" immune response or "protective" immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence and/or severity and/or duration of disease or any other manifestation of infection. For example, in representative embodiments, a protective immune response or protective immunity results in reduced viremia, whether or not accompanied by clinical disease. Alternatively, a protective immune response or protective immunity may be useful in the therapeutic treatment of existing disease.
An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both." Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 1 17 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host," Id. In some embodiments of this invention, methods are provided of treating a norovirus infection in a subject in need thereof by passive immunity, comprising
administering to the subject an effective amount of an antibody that specifically reacts with a norovirus (e.g., an antibody that binds a blockade epitope or motif of a GII.4 norovirus). Nonlimiting examples of such an antibody as described in the examples provided herein include human monoclonal antibody NBV 71.4 and mouse monoclonal antibody
GII.4.2002.G5. It would be understood that if the subject to be treated is a human, a humanized mouse monoclonal antibody (e.g., humanized GII.4.2002.G5) would be administered to the subject. The production of humanized monoclonal antibodies from non- human species is well known in the art, as are protocols for determining dosage and route of administration of such antibodies to a subject in an amount effective to treat a norovirus infection and/or protect a subject against the effects of norovirus infection.
A "subject" of the invention includes any animal susceptible to norovirus infection. Such a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.). In particular embodiments, the subject is a primate subject, a non-human primate subject (e.g. , a chimpanzee, baboon, monkey, gorilla, etc) or a human. Subjects of the invention can be a subject known or believed to be at risk of infection by norovirus. Alternatively, a subject according to the invention can also include a subject not previously known or suspected to be infected by norovirus or in need of treatment for norovirus infection. Subjects may be treated for any purpose, such as for eliciting a protective immune response or for eliciting the production of antibodies in that subject, which antibodies can be collected and used for other purposes such as research or diagnostic purposes or for administering to other subjects to produce passive immunity therein, etc.
Subjects include males and/or females of any age, including neonates, juvenile, mature and geriatric subjects. With respect to human subjects, in representative embodiments, the subject can be an infant (e.g. , less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or younger), a toddler (e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months), or a child (e.g., at least about 1, 2, 3, 4 or 5 years of age and/or less than about 14, 12, 10, 8, 7, 6, 5, or 4 years of age). In embodiments of the invention, the subject is a human subject that is from about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 12 to 18, 24, 36, 48 or 60 months of age, from about 18 to 24, 30, 36, 48 or 60 months of age, or from about 24 to 30, 36, 48 or 60 months of age.
A "subject in need" of the methods of the invention can be a subject known to be, or suspected of being, infected with, or at risk of being infected with, noro virus.
Pharmaceutical formulations (e.g., immunogenic formulation) comprising the norovirus epitopes, chimeric norovirus capsid proteins, polypeptides, chimeric norovirus VLPs, chimeric norovirus VRPs or chimeric norovirus particles, nucleic acids, vectors, cells or compositions of the invention and a pharmaceutically acceptable carrier are also provided, and can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (latest edition), in the manufacture of a pharmaceutical composition according to embodiments of the present invention, the composition of the invention is typically admixed with, inter alia, a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that is compatible with other ingredients in the pharmaceutical composition and that is not harmful or deleterious to the subject. The carrier may be a solid or a Hquid, or both, and is preferably formulated with the composition of the invention as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the composition. The pharmaceutical compositions are prepared by any of the well- known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients. In certain embodiments, the pharmaceutically acceptable carrier is sterile and would be deemed suitable for administration into human subjects according to regulatory guidelines for pharmaceutical compositions comprising the carrier.
Furthermore, a "pharmaceutically acceptable" component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are "undue" when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable components include any of the standard
pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents, typically provided as sterile compositions.
In some embodiments, the compositions of the invention can further comprise one or more than one adjuvant. The adjuvants of the present invention can be in the form of an amino acid sequence, and/or in the form or a nucleic acid encoding an adjuvant. When in the form of a nucleic acid, the adjuvant can be a component of a nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) and/or a separate component of the composition comprising the nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) of the invention. According to the present invention, the adjuvant can also be an amino acid sequence that is a peptide, a protein fragment or a whole protein that functions as an adjuvant, and/or the adjuvant can be a nucleic acid encoding a peptide, protein fragment or whole protein that functions as an adjuvant. As used herein, "adjuvant" describes a substance, which can be any immunomodulating substance capable of being combined with a composition of the invention to enhance, improve or otherwise modulate an immune response in a subject.
In further embodiments, the adjuvant can be, but is not limited to, an
immunostimulatory cytokine (including, but not limited to, GM CSF, interleukin-2, interleukin-12, interferon-gamma, interIeukin-4, tumor necrosis factor-alpha, mterleukin- 1 , hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules), SYNTEX adjuvant formulation 1 (SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline. Suitable adjuvants also include an aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes.
Other adjuvants are well known in the art and include without limitation MF 59, LT- K63, LT-R72 (Pal et al., Vaccine 24(6):766-75 (2005)), QS-21 , Freund's adjuvant (complete and incomplete), aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn - glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryi lipid A, trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80 emulsion.
Additional adjuvants can include, for example, a combination of monophosphoryi lipid A, preferably 3-de-O-acylated monophosphoryi. lipid A (3D-MPL) together with an aluminum salt. An enhanced adjuvant system involves the combination of a monophosphoryi lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in PCT publication number WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in PCT publication number WO
96/33739. A particularly potent adjuvant formulation involving QS21 3D-MPL & tocopherol in an oil in water emulsion is described in PCT publication number WO 95/17210. In addition, the nucleic acid compositions of the invention can include an adjuvant by comprising a nucleotide sequence encoding the antigen and a nucleotide sequence that provides an adjuvant function, such as CpG sequences. Such CpG sequences, or motifs, are well known in the art.
An adjuvant for use with the present invention, such as, for example, an
immunostimulatory cytokine, can be administered before, concurrent with, and/or within a few hours, several hours, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 days before and/or after the administration of a composition of the invention to a subject.
Furthermore, any combination of adjuvants, such as immunostimulatory cytokines, can be co-administered to the subject before, after and/or concurrent with the administration of an immunogenic composition of the invention. For example, combinations of
immunostimulatory cytokines, can consist of two or more immunostimulatory cytokines, such as GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules. The effectiveness of an adjuvant or combination of adjuvants can be determined by measuring the immune response produced in response to administration of a composition of this invention to a subject with and without the adjuvant or combination of adjuvants, using standard procedures, as described herein and as known in the art.
In embodiments of the invention, the adjuvant comprises an alphavirus adjuvant as described, for example in U.S. 7,862,829.
Boosting dosages can further be administered over a time course of days, weeks, months or years. In chronic infection, initial high doses followed by boosting doses may be advantageous.
The pharmaceutical formulations of the invention can optionally comprise other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, diluents, salts, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanol amine oleate, etc.
For injection, the carrier will typically be a liquid. For other methods of
administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and is typically in a solid or liquid particulate form.
The compositions of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical composition according to the invention, the VLPs are typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is optionally formulated with the compound as a unit-dose formulation, for example, a tablet. A variety of pharmaceutically acceptable aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N J.), and the like. These compositions can be sterilized by conventional techniques. The formulations of the invention can be prepared by any of the well-known techniques of pharmacy.
The pharmaceutical formulations can be packaged for use as is, or lyophilized, the lyophilized preparation generally being combined with a sterile aqueous solution prior to administration. The compositions can further be packaged in unit/dose or multi-dose containers, for example, in sealed ampoules and vials. The pharmaceutical formulations can be formulated for administration by any method known in the art according to conventional techniques of pharmacy. For example, the compositions can be formulated to be administered intranasally, by inhalation (e.g., oral inhalation), orally, buccally (e.g. , sublingually), rectally, vaginally, topically, intrathecal ly, intraocularly, transdermally, by parenteral administration (e.g. , intramuscular [e.g. , skeletal muscle], intravenous, subcutaneous, intradermal, intrapleural, intracerebral and intra-arterial, intrathecal), or topically (e.g., to both skin and mucosal surfaces, including airway surfaces).
For intranasal or inhalation administration, the pharmaceutical formulation can be formulated as an aerosol (this term including both liquid and dry powder aerosols). For example, the pharmaceutical formulation can be provided in a finely divided form along with a surfactant and propellant Typical percentages of the composition are 0.01-20% by weight, preferably 1-10%. The surfactant is generally nontoxic and soluble in the propellant.
Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, if desired, as with lecithin for intranasal delivery. Aerosols of liquid particles can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. Intranasal administration can also be by droplet administration to a nasal surface.
Injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one can administer the pharmaceutical formulations in a local rather than systemic manner, for example, in a depot or sustained-release formulation.
Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile formulation of the invention in a unit dosage form in a sealed container can be provided. The formulation can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can be from about 1 μ to about 10 grams of the formulation. When the formulation is substantially water- insoluble, a sufficient amount of emulsifying agent, which is pharmaceutically acceptable, can be included in sufficient quantity to emulsify the formulation in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.
Pharmaceutical formulations suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tables, as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water- in-oil emulsion. Oral delivery can be performed by complexing a compound(s) of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art. Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the protein(s) and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the pharmaceutical formulations are prepared by uniformly and intimately admixing the compound(s) with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or molding a powder or granules, optionally with one or more accessory ingredients. Compressed tablets are prepared by compressing, in a suitable machine, the formulation in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets are made by molding, in a suitable machine, the powdered protein moistened with an inert liquid binder.
Pharmaceutical formulations suitable for buccal (sub-lingual) administration include lozenges comprising the compound(s) in a flavored base, usually sucrose and acacia or tragacanth; and pastilles in an inert base such as gelatin and glycerin or sucrose and acacia.
Pharmaceutical formulations suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacterio stats and solutes, which render the composition isotonic with the blood of the intended recipient. Aqueous and non- aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of non -aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Pharmaceutical formulations suitable for rectal administration are optionally presented as unit dose suppositories. These can be prepared by admixing the active agent with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.
Pharmaceutical formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include, but are not limited to, petroleum jelly, lano ine, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. In some embodiments, for example, topical delivery can be performed by mixing a pharmaceutical formulation of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
Pharmaceutical formulations suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3:318 (1986)) and typically take the form of a buffered aqueous solution of the compound(s).
Suitable formulations can comprise citrate or bisNtris buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M active ingredient.
Further, the composition can be formulated as a liposomal formulation. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. The liposomes that are produced can be reduced in size, for example, through the use of standard sonication and homogenization techniques.
The liposomal formulations can be Iyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
The immunogenic formulations of the invention can optionally be sterile, and can further be provided in a closed pathogen-impermeable container. In embodiments of the invention, the dosage of a vims particle of this invention can be in a range of about 10 to about 10 plaque forming units (PFUs), In embodiments of this invention, the dosage of a VLP of this invention can be in a range of about 5 micrograms to 5 milligrams. In embodiments of this invention, the dosage of a protein of this invention can be in a range of about 10 to about 105 micrograms +/- adjuvant.
EXAMPLES
Noroviruses are ~38 nm icosahedral viruses with a ~7.5 Kb single-stranded, positive- sense RNA genome that encodes three large open reading frames (ORFs). ORF1 encodes the non-structural proteins, ORFs 2 and 3 encode the major and minor capsid proteins, respectively. Expression of the major capsid protein (ORF2) in Venezuelan equine encephalitis (VEE) virus or baculovirus results in the formation of virus-like particles (VLPs) composed of 90 copies of the major capsid protein dimer [11]. Noroviruses are grouped by the amino acid sequence of the major capsid protein. Viruses with less than 14.3% difference are classified as the same strain, 14,3-43.8% the same genotype, and 45-61.4% the same genogroup [12]. Currently, noroviruses are grouped into five genogroups (GI-GV).
Genogroups GI and GII are responsible for most human infections and are further subdivided into 8 and 21 different genotypes, respectively [1, 12].
Structurally, the capsid monomer is divided into three domains. The shell domain (S) forms the core of the particle and the protruding domain (P) extends away from the core. The P domain is further subdivided into the PI subdomain (residues 226-278 and 406-520) and the P2 subdomain (residues 279-405) [1 1]. The P2 subdomain is the most exposed region of the viral particle and it is well positioned to interact with potential neutralizing antibodies and histoblood group antigen (HBGA) ligands [13-17]. Previous studies have shown that the P2 subdomain of the major capsid protein of GI1.4 strains is evolving rapidly, resulting in new epidemic strains with altered carbohydrate ligand binding properties and antigenicity [13, 18- 23],
For the past two decades, the majority of norovirus outbreaks have been caused by strains within the genogroup II, genotype 4 (GII.4 strains) subcluster. Between 1995 and 2006, four major norovirus pandemics associated with GII.4 strains were characterized using molecular epidemiologic methods. During the mid-1990s [24] strain US95/96 was responsible for -55% of the norovirus outbreaks in the USA and 85% of the outbreaks in the Netherlands [25]. In 2002, the US95/96 strain was replaced by the Farmington Hills strain [26], which was associated with -80% of norovirus outbreaks [27] in the USA. In 2004, the Hunter GII.4 variant was detected in Australia, Europe, and Asia [28-30]. Hunter strains were largely replaced in 2006 by two new co-circulating GII.4 variants in the USA and Europe; Laurens (2006a) and Minerva (2006b) [5, 29, 31]. In 2009 GII.4.2009 New Orleans [1, 32] emerged and replaced Minerva, followed in 2012 by pandemic circulation of
GII.4.2012 Sydney [33].
A variety of studies using time ordered human outbreak sera and mouse monoclonal antibodies support the hypothesis that the GII.4 noroviruses are undergoing antigenic variation and that this variation contributes to the emergence of new outbreak strains over time [13, 22, 34-36]. However, the lack of a cell culture or small animal model for human norovirus cultivation restricts study of neutralization antibodies and epitopes. To circumvent this problem, highly informative in vitro assays have been developed that measure the ability of an antibody to "block" binding of a VLP to a carbohydrate ligand [13, 17, 37, 38], This assay is highly sensitive, as it differentiates between norovirus strains too similar to be distinguished by EIA. The clinical relevance of the blockade assay, as a surrogate
neutralization assay, has been confirmed in both infected chimpanzees [39] and Norwalk virus-infected humans [10, 40], Using human norovirus outbreak sera, VLP-immunized mouse sera and mouse mAbs [13, 35, 36], the early GII.4 strains (1987 and 1997) were antigenically indistinguishable from each other by EIA and surrogate neutralization assays. VLPs of strains circulating post 2002 had significantly less reactivity with sera directed against earlier strains and no reactivity to mouse mAbs directed to GIL4.1987. Conversely, select mouse mAbs generated against GII.4.2006 reacted with VLPs that circulated only from 2002 or later. No blockade antibodies were found to be in common between GII.4.1987 and GII.4.2006.
To identify evolving GII.4 blockade antibody epitopes, the evolution of the GII.4 noroviruses was assessed over a 36-year period of time by comparing strains from 1974 to 2010. In comparing these sequences, sites of variation in the P2 subdomain were noted, and these sites were mapped onto the crystal structure of the P-domain dimer for the 1997 strain VA387. Surface-exposed sites of variation were then examined to determine which residues may be close enough to constitute a single epitope, and five epitopes were predicted based upon this variation [17, 41],
Epitope A encodes significant amino acid changes over time and has also been demonstrated to be an evolving GII.4 blockade epitope using mouse mAbs [42]. Epitope A is conformational and is located on the top of the capsid proximal to the HBGA binding pocket. Six variable sites were close to each other in the region of this putative epitope, suggesting that these residues may work in concert to change the local structure of Epitope A. The variable, surface-exposed residues include amino acid positions: 294, 296-298, 368 and 372. In the present invention, additional residues at amino acid positions 292, 295, 357, 365, and 373 have been identified as residues of Epitope A. Epitope A is continuing to evolve in extant strains, whereby the amino acid at position 294 seems to vary extensively in strains from 2008-2010 (amino acid replacements P294A, P294S and P294T have been observed at this position).
Epitope B was identified based upon two variable residues at positions 333 and 382. While these residues are buried in the dimer interface between two chains, the patterns of variation at these sites suggest that they play an important role in the evolution of novel strains, perhaps by evolving replacements that allow the more surface exposed residues in other surface exposed epitopes to dramatically change the physiochemical properties of the amino acid replacements.
Residues 340 and 376 make up the variable residues of putative Epitope C. This putative conformation dependent epitope is on the surface and lateral edge of the capsid and is directly proximal to the HBGA binding pocket, suggesting that this epitope may play a role in receptor switching along with Epitope D.
Epitope D was initially characterized as comprising the three variable residues from positions 393-395. In the present invention, Epitope D has been identified as further comprising amino acid residues at positions 391, 396 and 397. In the first reported crystal structure for the GII.4 noroviruses, this region was reported to be a secondary HBGA binding site [16]. However, the location of this epitope on the surface of the capsid, directly proximal to the HBGA binding site, suggests that it likely plays a role in both receptor switching and in escape from herd immunity and perhaps both, simultaneously [13, 21, 43, 44]. Epitope D is close enough to the HBGA binding pocket to contribute to or inhibit carbohydrate binding, and yet variable enough to suggest that it is targeted by the immune response.
Putative Epitope E comprises variable residues 407, 412 and 413, which are surface exposed regions lateral to the HBGA binding pockets and other epitopes. In the present invention, residues at amino acid positions 355, 356, 357, 359, 364, 365 and 404 have been identified as being included in Epitope E. The residues 407, 412 and 413 vary with every major epidemic strain after 2002, suggesting that it is a hot spot for the emergence of immunologically novel GII.4 strains. Epitope E is a GIL4.2002 blockade antibody epitope [45], The fact that this putative epitope is lateral to the HBGA binding pockets suggests that antibodies are targeting regions interior to the capsid surface, which suggests that other epitopes may be present in the PI subdomain. A few variable residues do not necessarily identify the boundaries of a putative epitope. Moreover, it is nearly impossible to predict the surface area of a putative epitope by sequence analysis alone.
In the studies of this invention, both human and mouse anti-No V mAbs have been used, coupled with molecular biology approaches to exchange epitopes between GII.4 strain backbones to identify evolving blockade epitopes between GII.4 strains that have circulated from 1987 until 2009, Using this novel approach, three evolving blockade GII.4 antibody epitopes [17, 42, 45] have been identified. Epitope A comprises amino acids 292, 294, 295, 296, 297, 298, 357, 365, 368, 372 and 373 and is a highly variable blockade epitope that changes with new GII.4 strain emergence. Monoclonal human and mouse antibodies against Epitope A have been described as highly strain selective, lacking reactivity to other GIL4 strains chronologically removed from the immunizing strain. GII.4.2006 and GII.4.2009 were found to differ in reactivity to a human mAb targeting Epitope A, establishing a difference in human neutralizing epitopes between these two strains and supporting escape from herd immunity by antigenic variation at neutralizing epitopes as a mechanism for new NoV strain emergence.
Epitope D (residues 391, 393, 394, 395, 396 and 397) has also been confirmed as an evolving blockade epitope using human anti-No V mAbs. These findings are particularly interesting as Epitope D has been shown to modulate HBGA binding of GII.4 strains supporting the suggested correlation between epitope escape from herd immunity and altered HBGA binding [13 ].
Mouse mAbs have confirmed Epitope E (amino acids 355, 356, 357, 359, 364, 365, 404, 407, 412 and 413) as a GII.4.2002 Farmington Hills-specific blockade epitope [45].
Comparing reactivity of polyclonal sera collected from immunized mice and infected humans suggested antigenic variation within the GO.4 noroviruses [13, 35]. The development of mouse mAbs to different time-ordered GII.4 VLPs has greatly facilitated progress towards understanding the complex antigenic relations between these strains by clearly demonstrating antigenic variation over time and epidemic strain [17, 34, 36, 42, 47]. However, to maximally define the mechanistic relationships that exist between antigenic variation, immunity and HBGA binding patterns noted in the GII.4 noroviruses in the context of natural infection history, the cross reactivity patterns, blockade responses, and epitope targets of human anti- GII.4 monoclonal antibodies are needed. Robust approaches exist for the isolation of human monoclonal antibodies that are elicited following virus infection. Using human PBMCs as a source of memory B cells, a panel of human mAbs directed against GII.4 strains was created and used to examine the reactivity of these mAbs to a panel of time-ordered GII.4 VLPs using EIAs and surrogate neutralization assays. We identified one unique, broadly cross reactive antibody that differentially blocks GII.4, 1987 through 2009 VLP interactions with carbohydrate ligands, a potential immunotherapeutic for the treatment of acute or chronic GII.4 disease [17]. Unique antibody interactions were also defined with two different surface exposed epitopes that evolve over time. Importantly, antigenic variation in one of these epitopes correlated with changing carbohydrate ligand binding patterns over time, supporting the proposed relationship between epitope escape from human herd immunity and changing HBGA usage for virus docking [13], In addition to defining the first human monoclonal antibodies with therapeutic potential for treating acute and chronic NoV GII.4 infections, these data support the hypothesis that GII.4 norovirus evolution results in antigenic drift of neutralizing epitopes and consequently, antibody-driven HBGA receptor switching; thus, protective herd immunity is a driving force in norovirus evolution. EXAMPLES
Example 1. Identification of antigenic epitopes in GII.4 noroviruses
Table 1 lists several NoV strains of this invention. The evolutionary profile of the epitopes of various human NoV strains is shown in FIG 1. Five important antigenic epitopes in GII.4 noroviruses were identified. The differences between the epitope amino acid sequences in GII.4-1987 and GIL4-2006 are shown in Table 2. Three of these, epitopes A, D, and E, have been confirmed as GII.4 blockade epitopes. Epitope A is at positions 292, 294, 295, 296, 297, 298, 357, 365, 368, 372 and 373, Epitope B is at positions 333 and 382, Epitope C is at positions 340 and 376, Epitope D is at positions 391, 392, 393, 394, 395, 396 and 397 and Epitope E is at positions 355, 356, 357, 359, 364, 365, 404, 407, 412 and 413.
To determine if a VLP with blockade epitopes from multiple GIL4 strains could be constructed that could induce a broadly blocking immune response against several strains, mice were immunized with chimeric GII.4 V Ps and the sera were used to test the immune response. GII.4-2006.87A contains the A epitope from 1987 in the 2006 background. GII.4- 2006.87A.02E contains the A epitope from 1987 and the E epitope from 2002 in the 2006 background, as shown in FIG 2.
Mice were immunized with 10 ul (1 x 104) VRPs expressing different parental GII.4 capsids or chimeric GII.4 capsids (FIG. 3). Mice were immunized with VRPs and boosted with the same VRPs on day 21. Seven days post-boost, mice were euthanized and serum was collected to use in ELIS A-based assays to determine serum blockade response and reactivity to various VLPs (FIGS. 4A-C).
FIGS, 4A-C show sigmoidal curves of the blockade data. The dotted line indicates 50% blockade, the point at which the sera are considered blockade sera. Lines that do not cross the 50% mark indicate the inability of the sera to block that particular VLP. The further left the curve is shifted, the more efficiently the sera blocked that particular VLP. These data indicate that both sera from mice immunized with either chimeric VLP (GII.4-2006.87A, 2006.87A.02E) or a trivalent mix of VLPs (GII.4-1987+2002+2006) are able to block VLPs both included in the chimera cocktail (1987, 2002, 2006) and those not included (1997, 2009).
Sera from mice immunized with a single parental VLP (GII.4-1987, 2002, 2006, or 2009) were not able to block all of the VLPs tested (FIGS. SA-D).
FIGS. 6A-C represent the concentration of serum at which each sample reaches a blockade response (crosses the dotted line in the curve graphs— this is the EC50 or 50% blockade). Dots below the line indicate a blockade response for that VLP. The lower the point is on the graph, the better the blockade response of the serum. Sera from mice immunized with either chimera (GIL4-2006.87A, GII.4-2006.87A.02E) or the multivalent 1987+2002+2006 cocktail (GII.4-87/02/06) were able to block all VLPs tested, although to varying degrees. Sera from mice immunized with a single parental strain VLP (GII.4-1987, 2002, 2006, 2009) could not induce a blockade response against all tested strains (FIGS. 7A- D). Dots above the line indicate a VLP that was not blocked even at the highest serum concentration tested (5%).
Table 3 depicts a summary of blockade responses of parental, chimeric and multivalent sera. These data show that monovalent VLP vaccines designed after strains circulating between 1987-2009 (GII.4) elicit poor cross protection to related strains.
Multivalent vaccines (multiple VLPs, or multiple V Ps encoding different NoV VLP) give broader responses. Importantly, a VLP has been designed with one or multiple time-ordered GII.4 epitopes that elicited a broadly neutralizing antibody response that captured more unique strains.
Example 2. Identification of a GII.4 norovirus conserved blockade epitope using human monoclonal antibodies
The identification of a GII.4 norovirus conserved blockade epitope using human monoclonal antibodies is shown in FIG. 8. Monoclonal antibodies NVB 71 and NVB1 11 were assayed for the ability to block VLP interaction with carbohydrate ligand. Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to PGM in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment). Error bars represent SEM.
Example 3. Characterization of antibody epitopes and development of chimeric VLPs
Bioinformatic approaches identified five putative antibody epitopes on the surface of GII.4 noroviruses that appeared to be evolving over time and to correlate with the emergence of new GII.4 outbreak strains. Predicted epitopes were expanded to include interacting amino acids within 8A. Epitope A, Epitope B, Epitope C, Epitope D, Epitope E and HBGA binding sites mapped onto the P domain dimer of GI1.4.2002.
Hu mAb PGM binding blockade activity against GII.4.1987 epitope-exchanged VLPs for NVB 114 (FIG. 9A) and Epitope A exchanged between GII.4.1987 and GII.4.2006 (FIGS. 9C and E). Sigmoidal curves were fit to the mean percent control binding calculated by comparing the amount of VLP bound to PGM in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard error of the mean. Mean EC50 for blockade of each GII.4.1987 exchange epitope (FIG. 9B) and Epitope A exchange VLPs (FIGS. 9D and F). * VLPs with EC50 values significantly different from the EC50 for each parental VLP.
Sequence alignments of GIL4-2006b compared to intra-host evolved isolates P.D1 and P.D302 (top) and GII.4-2009 compared to GII.4-2012 (bottom) (FIG. 10A). Grey residue positions represent evolving residues within epitope A, while black residue positions represent new positions that have been identified as being part of epitope A. FIG 10B is a structural model showing the location of previously-established epitope A residues (grey) and newly identified epitope A residues (black). EC50 blockade titers for human mAb NVB 43.9 against VLPs representing GII.4-2009, GII.4-2012, GII.4-2012.R373N, GII.4-2012.09A
(2012 backbone with previously-established epitope A changes), and GII,4-2012.09A.R373N (2012 backbone with previously-established epitope A changes plus an R373N change). * VLPs with EC50 values significantly different from the EC50 for GII.4-2009 (FIG. IOC).
NVB 97 PGM binding blockade activity against Epitope D exchange VLPs.
Sigmoidal curves were fit to the mean percent control binding calculated by comparing the amount of VLP bound to PGM in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard error of the mean (FIG. 11 A). Mean EC50 for blockade of each blocked VLP. * indicates VLPs with significantly different EC50 compared GII.4.2006 (FIG. 11B).
Pymol was used to model the polar interactions within residues 390-395 (FIGS. 12A- C) and interactions between these residues and surrounding residues (FIGS. 12D-F) for outbreak strain GII.4-2006b and intra-host evolved isolates P.D1 and P.D302. PD302 has a D391N change compared to both GII.4-2006b and P.D1. GII.4-2006b is shown in FIGS. 12A and D, P.D1 is shown in FIGS. 12B and E, and P.D302 is shown in FIGS. 12C and F. Residues 390-395 are shown in dark grey for GII.4-2006b and in light grey for P.D1 and for P.D302. Dotted lines represent structure-based predicted polar interactions. Dark residues represent positions that interact with HBGAs (FIGS. 12D-F).
VLPs engineered to contain variations of Epitope E were assembled to test the impact of the epitope and the non-epitope backbone on anti-GII.4-2002-G6 binding and
neutralization (FIG. 13A). Anti-GII.4-2002-G6 EIA reactivity to the Epitope E constructs. * indicates chimeric VLPs with reactivity significantly different from parental VLPs (FIG. 13B). Blockade of Epitope E constructs binding to PGM by anti-GIL4-2002-G6. The mean g/ml mAb needed to block 50% of GO.4 VLP binding to ligand is indicated by the line in the box. The upper and lower whiskers of the box represent the maximum and minimum values. * indicates VLPs with BT50 significantly different from GII.4-2002 (FIG. 13C).
Epitope E was defined by variable residues 407, 412 and 413 (406, 41 1 and 412 for GII.4.1987) and all residues within 8 angstroms of these sites, as these additional residues are likely to be impacted by the structural differences (driven by the variable sites) that contribute to mAb recognition differences. GII.4.1987 E epitope (FIG. 14A) differs from GII.4.2002 (FIG. 14B) at positions 406/407 and 355. The N to S difference at 406/407 likely alters the rotameric position of R41 1 in the GII.4.2002 VLP, allowing it to extend further from the surface. (FIG. 14C) GII.4.1987 and GII.4.2002 superimposed. Because R41 1 is more buried in 1987, the mAb likely cannot interact as strongly with this residue in the GII.4.1987 VLP. GII.4.2002 (FIG. 14D) differs from GII.4.2006 (FIG. 14E) at five positions in the expanded epitope (positions 355-357, 412 and 413). Superimposition suggests that R41 1 of GII.4.2006 is buried, and variation at 355-357 may alter key interactions involving H357 and D355. (FIG. 14F) GII.4.2006.N412T superimposed upon GII.4.2006. Two potentially important residues are R41 1, which is more surface exposed in GII.4-2006.N412T, and T412, which is buried (FIG. 14G). GII.4.2002 superimposed on GII.4-2006.N412T (FIG. 14H). R41 1 is nearly identical suggesting that N412T frees the R41 1 side chain to extend away from the surface where it likely interacts with the mAb. Resides that regulate R41 1 make up site 1 , and 72% of binding can be recovered with modifications to R411. Site 2 is the second site and is comprised of residues 355-357, and particularly D355 which adds negative potential to the second site.
Expanded epitopes include residues within 8 angstroms of the confirmed epitope signatures including residues validated in follow up studies (FIG. 15).
Amino acid conservation and variation were mapped onto the crystal structure of the GII.4.2004 P domain dimer, with Chain A (dark grey) and Chain B (light grey) shown, to identify a region on the side of the PI subdomain that was conserved among GII.4 epidemic strains. The carbohydrate binding pocket is located in the P2 subdomain where it is exposed on the surface of the VLP (FIG. 16A), whereas the conserved region was found on the side of the P domain dimer, distal to the binding pockets (FIG. 16B), The ERK motif (FIG. 16C, rotated 60° counterclockwise on the X-axis compared to FIG. 16B) is comprised of three charged amino acids that are found at positions 316, 484, and 493 in the conserved region that is predicted to interact with NVB 71.4. Variation at position 310 is proximal to the conserved region (FIG. 16B) and may regulate binding to this conserved site.
NVB 71.4 (FIGS. 17A and D), GIIA2002.G5 (FIGS. 17B and E) and NVB 71.4 Fab (FIGS. 17C and F) were assayed for ability to block carbohydrate ligand interaction of GII.4.2006 VLPs at room temperature (black circles) and 37 °C (black squares) and
GII.4.2006.ERK VLPs at room temperature (grey circles) and 37 °C (grey squares).
Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment) and the mean EC50 ^g/ml) titer for blockade calculated and compared. The fold change in EC50 titer was defined as the mean ECso at 37 °C compared to room temperature. * Mean EC50 blockade titer for GII.4.2006.ERK significantly different from the mean EC50 blockade titer for GII.4.2006 at the same temperature. Non-blockade VLPs were assigned an EC50 of 2X the upper limit of detection for statistical analysis and denoted by a data marker on the graph above the dashed line (assay upper limit of detection) for visual comparison. Error bars represent the SEM on sigmoidal fit curves and 95% confidence intervals on Mean EC50 graphs.
The ERK motif was mapped onto the crystal structure of GII.4.2004 to identify sites that may be interacting with the ERK motif (FIG, 18 A). The ERK motif is highly conserved among epidemiologically important GII.4 strains while residue 310 has evolved in the most recent GII.4 strains with global distribution (FIG. 18B). GII.4.2002.G5 (FIGS. 19A and B), and NVB 71 A Fab fragments, and NVB 71.4 IgG (FIG. 19B) were assayed for ability to block the interaction of VLPs with carbohydrate ligand at room temperature. Sigmoidal curves were fit to the mean percent control binding (percent of VLP bound to ligand in the presence of antibody pretreatment compared to the amount of VLP bound in the absence of antibody pretreatment) and the mean EC50 ^g/ml) titer for blockade calculated. The fold change in potency (EC50 titer) was defined as the ratio between mutant VLPs and wildtype VLP at room temperature (FIGS. 19A and B). The fold change in temperature sensitivity was defined as the change in ratio between mean EC 50 at 37 °C compared to room temperature for the mutant VLP compared to ratio at both temperatures for the wildtype VLP (FIG. 19A). * Mean EC50 blockade titer for mutant VLP significantly different from the mean EC50 blockade titer for wildtype VLP at the same temperature. Error bars represent 95% confidence intervals. Fold increase and fold decrease are indicated by light grey and dark grey, respectively (FIG. 19B).
Sequences from GII.4-1987, GII.4-2002, and GIL4-2006 were aligned and residues in blockade epitopes A, D, and E are shown in FIG. 2 A. The location of blockade epitopes A, D, and E on the capsid P2 dimer is shown in FIG. 2B. Chimeric VLP P2 dimers are shown in FIG. 2C. Differential Epitope A residues from parental strain GII.4-1987 (A294V, R297H, N298D, S368T, E372N) are inserted into the parental GII.4-2006 strain background to create chimeric VLP GII.4-2006.87A. Chimeric VLP GII.4-2006.87A.02E contains the same GII.4-1987 residue substitutions as well as Epitope E substitutions (N412T, V413G).
EC50 blockade titers from mice immunized against different GH.4 VLPs are shown in FIG.20. Sera from mice immunized against GII.4-1987, GII.4-2002, and GII.4-2006 block only a subset of VLPs tested from 1987-2012 (FIG. 20A). Sera from mice immunized with chimeric VLPs GIL4-2006.87A and GII.4-2006.87A.02E block all VLPs tested from 1987- 2012 (FIG. 20B). A schematic showing the chimeric VLP vaccine strategy utilizing blockade epitopes from multiple circulating GII.4 strains is shown in FIG. 20C.
A strategy for the design and reformulation of chimeric VLPs is shown in FIGS. 21- 22. Starting with the current predominant circulating GII.4 strain, (1) epidemiological studies identify sequence changes in epitope A in minor variants that arise in the population. (2) Using the sequences of these epitope A variants, a panel of VLPs representing this genetic diversity is built and monoclonal antibodies and polyclonal sera can be used to evaluate changes in antigenicity for each variant. (3) From these data, specific variants that are antigenically distinct from the predominant strain are identified and can be more closely monitored in the population. (4) If one of these variants seems likely to emerge as a new predominant strain, the chimeric VLP is already made and can quickly be introduced into a new vaccine formulation. (5) When a new predominant strain emerges, this process can be repeated in order to consistently address GII.4 antigenic variation. Example 4. Antibody Access to a Conserved GII.4 Norovirus Blockade Epitope is Regulated by Particle Conformation
Virus-like particles. Synthetically derived (Bio Basic INC, Amherst, NY) epitope- engineered or outbreak strain ORF2 genes were inserted directly into the VEE replicon vector for the production of virus replicon particles (VRPs), as described (40, 47). VLPs were expressed in BHK cells and purified by velocity sedimentation in sucrose followed by simultaneous concentration and dialysis into PBS using 100 kDa MWCO centrifugal filter units (Millipore), if needed. Bac-GII.4.2009 (New Orleans) VLPs were obtained from the Centers for Disease Control and Prevention in Atlanta, GA and were produced by expression in the baculovirus system and purified by cesium chloride gradient. VLP protein
concentrations were determined by the BCA Protein Assay (Pierce, Rockford, IL), Uranyl acetate stained VLPs were visualized by transmission electron microscopy (TEM).
Sequences used to produce VLPs were identified from stool samples from multiple infected individuals. Further, all VLPs bind to human mabs and saliva, synthetic HBGAs and pig gastric mucin.
Monoclonal Antibodies. The characteristics of the antibodies used in this study have been previously published except GII.4.2002.G5. Details are described in (52) for the human mabs and in (40, 74) for the mouse epitope A mabs, GII.4.2002.G5 is a mouse mab generated by hyperimmunization with GII.4.2002 VLPs, as described (41). This antibody is now commercially available from Maine Biotech (MAB227P). Fabs were obtained by papain cleavage using papain immobilized on beaded agarose resin (30 IU/mg) (Pierce) followed by HiTrap protein-A (GE Healthcare) and size-exclusion chromatography
(Superdex 200 from GE Healthcare).
Blocking Of Binding (BOB) Assay. For experiments using human polyclonal serum, human mabs were purified on protein A or G columns (GE Healthcare) and biotinylated using the EZ-lmk NHS-PEO solid-phase biotinylation kit (Pierce). The competition between polyclonal serum antibodies and biotinylated humabs for binding to immobilized VLPs (1 g/ml) was measured by EIA. Briefly, plasma samples were added to GII.4.1997 or
GII,4.2006-coated plates at different dilutions. After 1 hour, biotinylated humab was added at a concentration corresponding to 80% of the maximal OD level, and the mixture was incubated at room temperature for 1 hour. Plates were then washed with PBS-0.05% Tween- 20 and bound biotinylated humab was detected using AP-labeled streptavidin (Jackson Immunoresearch). The percentage of inhibition was tested in duplicates and calculated as follows: (H(OD sample-OD neg ctr) / (OD pos ctr-OD neg ctr)))xl00. BD80 value was calculated by interpolation of curves fitted with a 4-parameter nonlinear regression. For screening donor plasma samples and human mab blocking of binding of mouse mabs, the binding titers to respective coated VLPs were determined by EIA by measuring the dilution required to achieve 50% maximal binding (EC50) as previously described (52). EIA plates were coated at 0.25 μg ml VLP for human mab BOB of mouse mab assays.
Analyzing Epitopes. Representatives of the capsid amino acid capsid sequences of
Gil strains and GII.4 strains (28) from 1974 to 2012 were aligned using ClustalX version 2 (75) and the amino acid residues that were conserved among all GO capsid sequences and all GII.4 capsid sequences were mapped onto the GII.4.2004 (PDB accession: 3JSP) (65) crystal structure to identify areas that were conserved among all Gil and all GII.4 capsid proteins. The original analysis was performed using the crystal structure for GII.4.1997 as the distances in the structure used for making the epitope prediction would be more reliable than in a homology model. The ERK and EHNQ motifs were identified as regions that were conserved among Gil noro viruses, and highly conserved among GII.4 viruses. E316, R484, and K493 (ERK) and E488, H501, N522 and Q523 (EHNQ) were identified as conserved residues in these regions that carried a charge and had exposed side chains that protruded. These sites were targeted for mutagenesis using the rationale that preserving the charge of these residues would preserve the structural components necessary for VLP formation.
VLP-Carbohydrate Ligand-Binding Assay.
EIA plates were coated with 10 ^πιΐ Pig Gastric Mucin (PGM) for 4 hours and blocked over night at 4°C in 5% dry milk in PBS-0.05% Tween-20 before the addition of increasing concentrations of VLP. Bound VLP were detected by a rabbit anti-GII.4 norovirus polyclonal sera made from hyperimmunization with a cocktail of GII.4.1987, GII.4.2002, GII.4.2006 and Gil .4.2009 VLPs, followed by anti-rabbit IgG-HRP (GE Healthcare) and color developed with 1-Step Ultra TMB ELISA HRP substrate solution (Thermo-Fisher). Each step was followed by washing with PBS-0.05% Tween 20 and all reagents were diluted in 5% dry milk in PBS-0.05% Tween-20. All incubations were done at room temperature. PGM at 10 μg/ml is a saturating concentration and cannot distinguish carbohydrate affinities between VLPs but does give maximum binding potential of the entire panel of GII.4 VLPs. Half maximum binding (EC50) values were calculated using sigmoidal dose response analysis of non-linear data in GraphPad Prism 6. Percent of maximum binding was defined compared to the mean OD 450 nm of 12 μ^ηιΐ VLP.
VLP-Carbo hydrate Ligand-Binding Antibody Blockade Assay.
For blockade assays, PGM-coated plates were prepared as described above. VLPs (0.25 μ /ηιΙ) were pretreated with decreasing concentrations of test mab for 1 hour before being added to the carbohydrate ligand-coated plates for 1 hour. Wash steps and bound VLP were detected as described above. The percent control binding was defined as the binding level in the presence of antibody pretreatment compared to the binding level in the absence of antibody pretreatment multiplied by 100. Antibody- VLP and VLP -PGM incubations were done at room temperature or 37°C, as described for each figure. All other incubations were done at room temperature. Antibodies were tested for blockade potential against GIL VLPs at two-fold serial dilutions. Additional concentrations of purified antibodies were tested if needed to complete the sigmoidal dose-response curve. Blockade data were fit using sigmoidal dose response analysis of non-linear data in GraphPad Prism 6. EC50 values were calculated for antibodies that demonstrated blockade of at least 50% at the dilution series tested. Monoclonal antibodies that did not block 50% of binding at the highest dilution tested were assigned an EC50 of 2X the assay upper limit of detection for statistical comparison. EC 50 values between VLPs were compared using the One-way ANOVA with Dunnett posttest, when at least three values were compared or a student's T test when only two values were compared. A difference was considered significant if the P value was <0.05. Of note, VLP concentrations in blockade assays are in the low nanomolar range and therefore cannot discriminate between antibodies with sub-nanomolar affinities. Antibody- VLP interactions were validated for compliance with the law of mass action by preforming blockade assays of GII.4.1997 and GII.4.2006 at 0.25, 0.5, 1 and 2 μ^ιυΐ VLP. EC50 values for antibody blockade varied less than 2-fold (1 dilution) between any combination of VLP concentration tested, indicating that under the test conditions, antibody is in excess to the VLP and the tenants of the law of mass action are met for the antibody- VLP binding. Blockade assays using human type A or B saliva as the source of carbohydrate ligand were performed as described (76) with 0.5 μg/ml VLP at room temperature and 37°C.
Antibody relative affinity measurements. Antibody ¾ measurements were done as previously described (67) at room temperature and 37°C. Briefly, EIA plates were coated with 0.25 μg/ml VLP in PBS, blocked, and incubated with serial dilutions of test antibody. Bound antibody was detected by anti-human IgG-HRP and color developed as described above. ¾ values were calculated using one-site specific binding equation in GraphPad Prism 6. a values were validated by repeating the above assay at a range of VLP concentrations. There was less than a two-fold difference in calculated Kd for any concentration between 0.13 and 1.0 μg/Γml for any antibody tested confirming that the antibody was in excess of the VLP under test conditions.
VLP-Protein A gold staining. VLPs were incubated with 5 g/ml human mab followed by 1/100 dilution of Protein A conjugated to 10 nm gold particles, absorbed onto prepared grids, stained with 2% Uranyl acetate and visualized by TEM. Staining specificity was validated by counting fifty fields of the negative control (VLP minus human IgG plus Protein A-gold). Only one gold particle was observed near a VLP in the fifty negative control fields.
Antibodies to conserved NoV epitopes are rarely detected in human serum samples. Monoclonal Abs and polyclonal sera raised in response to mouse immunization or human infection have identified epitope A as the immuno-dominant component of the GII.4 antibody blockade response, accounting for 40-50% of total blockade activity in serum (47, 59). To estimate the fraction of antibodies specific for conserved GII.4 epitopes in the overall serum antibody response, one hundred serum samples collected from healthy individuals were assayed for ability to block binding of human mabs NVB 61.3 and NVB 71.4 in a blockade of binding (BOB) assay (60, 61). Both human mabs recognize a broad panel of antigenically-diverse, epidemiologically-significant GII.4 NoV strain VLPs by EIA but only NVB 71.4 is capable of blocking VLP-ligand interactions (52). When tested against GII.4.1997 (Figure 23 A) or GII.4 2006 (Figure 23B), three sera were able to compete with NVB 61.3 binding by more than 80% while the remaining did not show significant inhibition, in spite of variable binding to tested VLPs (Figure 23, right column of each panel). Eighteen serum samples competed with NVB 71.4 binding to GII.4.1997 and six sera could compete for binding to GII.4.2006 by more than 80% (Figure 23). These data indicate that antibodies to GIL4 conserved epitopes may be rare in human serum samples even if the binding titers to the tested VLPs were high for the majority of the sera (EC50 value reported in the right column of each panel).
The conserved GH.4 blockade epitope is likely not surface exposed and antibody access to the epitope is regulated by particle conformation. To characterize the epitope recognized by NVB 71.4 we began by comparing the profile for NVB 71.4 blockade of a time-ordered panel of GII.4 VLPs representing circulating GII.4 strains from 1987 through 2012. As shown previously (52), blockade curves had relatively shallow slopes (range 0.68- 0.92) (Figure 24A). These data suggest that access of NVB 71.4 to the conserved blockade epitope may be restricted under the test conditions. Therefore, we repeated the blockade assay for NVB 71.4 against the panel of GII.4 VLPs at 37°C to increase the probability of the VLPs adopting a conformation more favorable for antibody binding during the incubation time (62). Incubation at 37°C significantly increased the blockade capacity of NVB 71.4 for the panel of GIL4 VLPs (Figure 24B). Further, the blockade curves demonstrated steep slopes (range 1.1 -2.8) with complete blockade reached at antibody saturation for each VLP. In agreement with previous findings (52), NVB 71.4 did not block each GII.4 VLP equivalently at room temperature or 37°C. Incubation at the higher temperature resulted in significantly less antibody needed for blockade of GII.4.1987 (21.4-fold less), GII.4.1997 (35.4-fold less), GII.4.2002 (5.0-fold less), GII.4.2006 (10.5-fold less), GII.4.2009 (6.9-fold less) and GII.4.2012 (9.9-fold less) (Figure 24C). Incubation at 37°C did not broaden the number of strains blocked by NVB 71.4, as the higher temperature did not allow blockade of any non-GII.4 VLPs tested, in agreement with previous findings for blockade at room temperature (52). Temperature dependent NVB 71.4 blockade activity was confirmed with alternative ligand sources human type A and type B saliva. As demonstrated for PGM, all of the tested VLPs were blocked at lower concentrations of NVB 71.4 at 37°C compared to RT. Although the temperature effect was retained across ligand sources, the degree of temperature effect varied by both GII.4 VLP and between the three types of ligand, in agreement with other reports demonstrating that antibody blockade potency varies between different ligand sources. These results suggest that although NVB 71.4 recognizes a conserved blockade epitope, the epitope is composed of both conserved and variable residues within the GII.4 capsid and, further, that the effect of temperature on the particle may also impact particle binding in a ligand specific mechanism.
In comparison, EC 50 titers for blockade of surface epitopes A and D were only minimally impacted by temperature. GII.4.2006 blockade by human mabs that bind to surface-exposed epitopes A and D required 1.4 and 1.3-fold less antibody, respectively, for 50% blockade of binding at 37°C compared to room temperature (data not shown). Although the mean EC50 titers for blockade of epitopes A and D are significantly different between room temperature and 37°C, the fold difference between the values reflects less than one two- fold serial dilution. These data indicate that unlike epitopes A and D, the conserved blockade epitope recognized by NVB 71.4 may not be readily accessible on the viral particle at all times, resulting in regulated antibody access under tested conditions.
Mouse mabs support the findings indicating that human antibodies that recognize the conserved GII.4 blockade epitope are relatively rare (Figure 23). In our screen of over 100 mouse mabs against GII.4 VLPs, we have identified only one mab with broad GII.4 blockade activity and this cross-blockade was temperature dependent (Figure 25). In agreement with NVB 71.4 findings, GII.4.2002.G5 mouse mab did not block each GII.4 VLP equivalently at room temperature or 37°C. Incubation at the higher temperature resulted in less antibody needed for blockade of GII.4.1987 (46-fold less), GII.4.1997 (8.5-fold less), GII.4.2002 (9.2- fold less), GII.4.2006 (10.2-fold less), GII.4.2009 (3,6-fold less) and GII.4.2012 (3.9-fold less). Incubation at 3 °C did not broaden the number of strains blocked, as the higher temperature did not allow blockade of any non-GII.4 VLPs tested. The varied degrees of blockade between different GII.4 VLPs suggests that the epitope GII.4.2002, G5 recognizes is composed of both residues that are conserved and variable across the GII.4 panel, as observed for NVB 71.4 (Figure 24).
Viruses and virus-like particles are dynamic structures and the degree of structural flexibility is temperature sensitive in at least some cases (62, 63). Moreover, studies with flaviviruses have demonstrated that the source of virus particles (e.g., cell lines) and host expression patterns (e.g., furin proteases) may also alter particle conformation or dynamics (64). While this study is the first to show that VLPs produced from VEE replicons adopt different conformations, to our knowledge no studies have demonstrated that viruses or VLPs assembled in the baculovirus insect cell system which functions at 27-28°C, are similarly dynamic. Therefore, we compared GII.4.2009 VLPs produced in the baculo virus-based insect system (27°C) and the VEE-based mammalian system (37°C) for antibody blockade at room temperature and 37°C. importantly, the primary nucleotide sequence of both
GII.4.2009 capsid constructs is identical (GenBank accession number ADD10375) in both constructs. For both mammalian and insect cell-produced GII.4.2009 VLPs, blockade of surface epitope A was efficient and not temperature sensitive (< 1.3-fold less antibody needed for 50% blockade) (Figure 26A). Unexpectedly, NVB 71.4 blockade was also not temperature sensitive (1.3-fold more antibody at 37°C) for the insect cell-produced VLPs, compared to 6.9-fold less antibody needed at 37°C for the mammalian cell produced VLP, Further, NVB 71.4 blockade of GII.4.2009 VLP produced in insect cells required 29.3-fold less antibody for 50% blockade at room temperature and 3.2-fold less at 37°C compared to GII.4.2009 VLPs produced in mammalian cells (0.1 133 and 0.1503 μ^ιηΐ compared to 3.322 at room temperature and 0.4817 μg/ml at 37°C (Figures 26B and 24 and (52)).
GII.4.2002.G5 needed 93-fold less antibody at room temperature and 19-fold less at 37°C for 50% blockade for GII.4.2009 VLPs produced in insect cells compared to mammalian cells (0.0843 and 0.1173 μg ml compared to 7.8 μg/ml at room temperature and 2.177 μ§/πύ at 37°C (Figures 26C and 25). This lack of temperature effect on Bac-GII.4.2009 blockade was maintained when B saliva was used as the Hgand source and when NVB 71.4 Fab fragments were used for the blocking antibody. These data support other study findings suggesting that factors outside of the capsid sequence can modify VLP antigenicity in subtle ways and support the hypothesis that antibody access to the conserved GII.4 blockade epitope is regulated by temperature and likely particle conformation.
Identification of a conserved GII.4 motif with epitope-like features. Using the crystal structure of GII.4.2004 P domain dimer (PDB accession: 3JSP) (65), conserved and variable amino acids were mapped onto the P domain dimer surface. A region that was highly conserved among GII.4 norovirus strains was identified on the side of the P domain dimer (Figures 16A and B), within the PI subdomain, interior to the exposed surface of the P2 subdomain, and distal to the carbohydrate binding pockets that correlate with binding differences to NVB 71.4 (Figures 16B and C). This region contained several conserved amino acids in an area large enough to represent a potential antibody binding site (>10002 A), including charged amino acids at positions E316, R484, and K.493 (post- 1997 GII.4 numbering) (Figure 16C). These amino acids were named the ERK motif (Figure 16C). The ERK motif is highly conserved among GII.4 strains that circulated between 1987 and 2012 and was predicted to be either a binding site for or a regulator of NVB 71.4 binding. In addition, amino acid position 310 was identified as a site of variation among contemporary GII.4 epidemic strains (2009 and 2012) that was proximal to the highly conserved region containing the ERK motif (Figure 16D).
Conservation of the ERK motif and its sub-surface PI location indicated that changes in these residues could be detrimental to viral particle structure or stability. Therefore, to evaluate the impact of the ERK motif on antibody blockade activity we designed mutant VLPs in the GII.4.2006 backbone that conserved the residue charge but changed the residue side chain length. The GII.4.2006.ERK clone contains substitutions E316D, R484K and K493R (Figure 27A). For comparison, we designed an additional PI domain mutated VLP based on a conserved GII antibody epitope recently published (66). GII.4.2006.EHNQ contains mutated residues E488D, H501K and N522Q and Q523N (post-1997 GII.4 numbering) (Figure 27A). The GII,4.2006,ERK substitutions did not notably alter particle structure as measured by electron microscopy visualization and ligand binding ability;
however, the GII.4.2006.EHNQ substitutions resulted in miss-folded viral particles that were unable to bind carbohydrate ligand (Figures 27B-C), emphasizing the challenge of studying non- surface exposed epitopes where even conserved residue changes can alter or even compromise particle integrity.
The GII.4 conserved ERK motif impacts NVB 71.4 and GH.4.2002.G5 blockade capacity with little impact on temperature sensitivity. As the substitutions made within GIL4.2006.ERK retained ligand binding activity, we evaluated the impact of these residue changes on the blockade potency of NVB 71.4, GII.4.2002.G5, and antibodies to surface exposed epitopes. ERK substitutions resulted in minimal increases in blockade ability for both epitope A and D antibodies (1.3-fold less antibody needed for 50% blockade at 37°C compared to room temperature for both human mabs, data not shown). However, the ERK motif substitutions resulted in complete loss of blockade potency of NVB 71.4 at room temperature. Blockade potency was restored at 37°C (2.519 μg/ml)! although significantly more antibody was needed for blockade compared to GII.4,2006 (4.1 -fold more antibody) (Figure 17A). Similarly, GIL4.2002.G5 did not block GII.4.2006.ERK at room temperature but gained limited blockade potency at 37°C (11.43 μg ml) (Figure 17B). However.
significantly more antibody was needed for blockade of GII.4.2006.ERK compared to
GII.4.2006 even at the elevated temperature (3.6-fold more). Further, blockade of GII.4.2006 and GII.4.2006.ERK with NVB 71.4 Fab fragments was more potent (lower EC50 value) but similarly temperature sensitive compared to NVB 71.4 IgG. Notably, the EC50 values were 2.1-fold different at room temperature (1.758 verses 0.8052) and 1.4-fold different at 37°C (0.1807 compared to 0.1259), indicating that with the smaller epitope-binding molecule, the ERK residues do not effect antigenicity (Figure 17C), Further, ERK substitutions negatively impact blockade potency for both conserved epitope antibodies but do not negate the compensatory effect of incubating at higher temperature, indicating that the ERK residues may be affecting antibody access to the epitope instead of the antibody binding strength for the epitope.
Quantitative EIAs (67) further indicate that ERK residue substitutions do not affect antibody affinities. Based on the differences in blockade titer, if the ERK substitutions were primarily affecting antibody affinity we would expect a 10-fold change in functional affinity for NVB 71.4 at room temperature and a 4-fold change at 37°C. However, there is less than a two-fold difference (one serial dilution) between antibody functional affinities (¾ values) of NVB 71.4, GII.4.2002.G5 and epitope D human mab for GII.4.2006 and GII.4.2006.ERK VLPs between room temperature and 37°C (Table 4). The lack of variation in ¾ values is contrary to findings for in-solution blockade assay EC50 values and suggests that when VLPs are immobilized on plastic plates antibody access to the conserved GII.4 blockade epitope is no longer restricted, further indicting that particle conformation regulates antibody access to the conserved blockade epitope.
Residue 310 modulates antibody blockade potency and temperature sensitivity.
In comparison to the highly conserved ERK motif, position 310 has recently begun to vary. Dominant GII.4 strains circulating between 1987 and 2006 conserved an asparagine at 310. With the emergence of GII.4.2009, N310 became S310. Subsequently, GII.4.2012 replaced the serine at 310 with an aspartic acid (Figure 18B). To investigate the role of residue 310 in GII.4 VLPs, we first developed mutated VLPs that exchanged the 310 residue between two additional GII.4 backbone sequences (Figure 28A). These substitutions did not notably alter particle structure as measured by electron microscopy visualization and ligand binding ability (Figures 28B-C) or blockade by epitope A or D human mabs. We exchanged the S310 residue found in GII.4.2009 with the D310 residue found in GII.4.2012 creating
GIL4.2009.S310D and GII.4.2012.D310S VLPs. In these constructs the ERK motif was unchanged. We then evaluated the impact of residue 310 on NVB 71.4, NVB 71.4 Fab and GIL4.2002.G5 blockade potency and temperature sensitivity (Figure 19). For both NVB 71.4 and GII.4.2002.G5, exchange of residue 310 between GII.4.2009 and GII.4.2012 resulted in an exchange of potency and temperature sensitivity phenotypes (Figures 19A and B). GII.4.2009.S310D required more antibody for blockade (2 and 4.1 -fold more) at room temperature and was 10.5 and 12-fold more temperature sensitive for NVB 71.4 and
GII.4.2002.G5, respectively. Conversely, GII.4.2012.D310S blockade potency increased 2.7 and 3.2-fold and temperature sensitivity decreased 4.6 and 3.1 -fold for each antibody. NVB 71.4 Fab had modestly increased potency at room temperature (1-2.8 fold) for the 310 mutant VLPs and the blockade was less temperature sensitive (2.4-8.0 fold) compared to wildtype, indicating that the smaller molecule has better access to the epitope.
To evaluate the interplay between residue 310 and the ERK motif, we created VLP
GII.4.2009.ERK containing both the S310D and ERK substitutions (S310D, E316D, R484K and K493R) (Figure 28 A). This VLP is called ERK, instead of SERK because of the asparagine found at 310 in the GII.4 VLPs from 1987-2006. Interestingly, combining the 310 and ERK residue changes in the GII.4.2009 backbone resulted in a VLP that was similarly blocked as GII.4.2009 for NVB 71.4 but required 4.1 -fold more GO.4.2002.G5 for 50% blockade. Of note, for both IgGs and NVB 71.4 Fab the ERK substitutions reduced the advantage of incubating at higher temperature by ~50% compared to wildtype VLP blockade. As there was less than a 2-fold difference in ¾ values for NVB 71.4 or GII.4.2002.G5 binding to GII.4.2009 and GIL 4.2009. ERK at room temperature or 37°C, it is unlikely that ERK forms the antibody epitope but instead that 310 and the ERK residues together form a regulating network. Blockade by anti-epitope A and D human mabs was unaffected by the 310 or ERK residue mutations indicating that the substitutions were specifically targeting the conserved blockade epitopes and not causing global particle disturbances. These data indicate in multiple Gil .4 backbones that residue 310 has a subtle effect on blockade potency at room temperature and a more significant effect on temperature sensitivity of the conserved blockade epitope, Comparison of the effect of serine verses aspartic acid at position 310 indicates better access to the epitope because of variation in regulating residues reduces the effect of incubating at higher temperature.
NVB 71.4 VLP-ligand interaction blockade is not explained by particle disassembly or steric hindrance. The mechanism of antibody blockade of VLP binding to carbohydrate ligand is unknown. To further explore the mechanisms of NVB 71.4 blockade we stained GII.4.2009 and GII.4.2009.ERK VLPs with NVB 71.4 and epitope A human mabs and Protein A gold particles and observed antibody labeled VLPs by negative stain electron microscopy (Figure 29). Both NVB 71.4 and the epitope A human mabs labeled intact VLPs, indicating that the antibody-induced lack of ligand binding was not the result of antibody-mediated particle disassembly or that NVB 71.4 preferentially binds to
disassembled capsid protein. To evaluate if NVB 71.4 binding to sub-surface sites altered the particle surface in a way that was undetectable by EM but rendered the particle unamenable to interactions at the surface, antibody blockade of binding competition assays were performed using antibodies to surface-exposed, conformation-dependent epitope A and subsurface, conformation-dependent NVB 71.4 (Figure 30). When VLP coated plates were pre- incubated with an epitope A human mab, binding of a mouse epitope A mab was reduced. The epitope A human mab blocked 50% of binding of a mouse epitope A mab at 0.7325 μ^πιΐ for GII.4.1997 and 0.1419 μ^ηιΐ for GII.4.2006. Binding of the epitope A human mab did not affect binding of the mouse mab GII.4.2002.G5 for either VLP. Likewise, a strain mismatched epitope A human mab did not affect binding of either the mouse epitope A or GII.4.2002.G5 antibody binding for either VLP. Conversely, pre-incubation of the VLP with NVB 71.4 did not affect binding of the mouse epitope A mabs but decreased binding of Gil A2002.G5. NVB 71.4 human mab blocked 50% of binding of mouse GII.4.2002.G5 at 0.0982 μ^ηιΐ for GII.4.1997 and 0.1913 ^ηιΐ for GII.4.2006. Combined, these data indicate that VLPs bound by NVB 71.4 retain conformation and spatial flexibility for interaction with molecules that bind to the particle surface, suggesting neither particle disassembly nor steric hindrance is likely to explain NVB 71.4 blockade activity FIG 31 shows the mapped epitopes and NERK motif of GII.4 noroviruses. The previously described evolving antibody blockade epitopes A-E are shown on the surface next to the carbohydrate binding sites (circled) (Panel A). The NERK motif is distal to the carbohydrate binding sites (denoted by arrows) (Panel B).
FIG. 32 shows a proposed model for regulation of antibody access to the conserved
GII.4 blockade epitope(s) by the ERK motif and VLP structural conformation.
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.
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Zakikhany, ., et al., Molecular Evolution of GII-4 Norovirus Strains. PLoS ONE, 2012. 7(7); p. e41625. Table 1. NoV strains
VLP Pandemic GenBank Original Strain SEQ
Cluster Access. No, ID NO
GIL.4.1974 ACT76139.1 1
GII.4.1987 Ancestral AA 50355.1 Hu/NLV/GII MD 14512/1987/US 2
GII.4.1997 US95/96 AFJ04707.1 Outbreak Isolate 3
GII.4.1997 AF 75854.1 14
GII.4.2002 Farmington Hills AFJ04708.1 Outbreak Isolate 4
GII.4.2002a n/a 5
GII.4.2004 Hunter AAZ31376.2 Hu/GII.4/Himter284E/04O/AU 6
GII.4.2005 None BAE98194.1 Hu/Sakai/04/179/2005/JP 7
GII.4.2006 Minerva 2006b AFJ04709.1 Outbreak Isolate 8
GII.4.2007 AB496912.1 Hu/GII.4/cruiseship/2007/ZAF 9
GII.4.2008a ACX31885.1 Hu/GII.4/NSW390I/AU 10
GII.4.2008S BAH30707.1 Hu/GII.4/Stockholm/19865/SE 11
GII.4.2009 Minerva 2006b ADD10375.1 Hu/GII.4/New_Orleansl 805/2009 USA 12
GII.4.2012 Sydney AFV08795.1 Hu/GII.4/Sydney NSW0514/2012/AU 13
Attorney Docket No. 5470-659WO ble 2. GII.4-1987 and GII.4-2006 epitope differences.
Figure imgf000056_0001
# Epitope E for GII.4-1987 is at amino acid positions 406, 411 and 412
Table 3. Blockade responses of parental, chimeric and multivalent sera.
Figure imgf000056_0002
TABLE 4 Monoclonal antibody functional affinities for Gil .4.2006 and GII.4.2006.ERK at room temperature and 37°C
mab Temp GII.4.2006 Kd GII.4.2006.ERK ¾ (nM)
(°C) (nM)
NVB 71.4 T 0.48 0.56
NVB 71.4 37 0.27 0.29
GII.4.2002.G5 RT 0.87 2.0
GIL4.2002.G5 37 0.57 0.87
Epitope D RT 0.78 0.99
Epitope D 37 0.39 0.41

Claims

THAT WHICH IS CLAIMED IS:
1. A chimeric norovirus capsid protein comprising a norovirus VP1 major capsid protein backbone comprising a norovirus epitope and/or motif selected from the group consisting of:
a) Epitope A (amino acids at positions 292, 294, 295, 296, 297, 298, 357, 365, 368, 372 and 373 of the amino acid sequence of SEQ ID NO:4);
b) Epitope B (amino acids at positions 333 and 382 of the amino acid sequence of SEQ ID NO:4);
c) Epitope C (amino acids at positions 340 and 376 of the amino acid sequence of SEQ ID NO:4);
d) Epitope D (amino acids at positions 391, 393, 394, 395, 396 and 397 of the amino acid sequence of SEQ ID NO:4);
e) Epitope E (amino acids at positions 355, 356, 357, 359, 364, 365, 404, 407, 412 and 413 of the amino acid sequence of SEQ ID NO:4);
f) NERK motif (amino acids at positions 310, 316, 484 and 493 of the amino acid sequence of SEQ ID NO:4); and
g) any combination of (a) through (f) above,
wherein the norovirus epitope and/or motif is from a norovirus strain that is different from the norovirus VP1 major capsid protein backbone,
2. The chimeric norovirus capsid protein of claim 1 , wherein the norovirus VP1 major capsid protein backbone is from a genogroup Gil, genotype 4 (GIL 4) strain.
3. The chimeric norovirus capsid protein of claim 2, wherein the norovirus VP1 major capsid protein backbone is from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002 GIIA2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GIL4.2008a, GII.4.2009 or GII.4.2012.
4. A synthetic backbone molecule comprising a norovirus epitope and/or motif selected from the group consisting of:
a) Epitope A;
b) Epitope B;
c) Epitope C; d) Epitope D;
e) Epitope E;
f) NER motif; and
g) any combination of (a) through (f) above,
wherein the backbone molecule allows for formation of a conformational epitope.
5. A norovirus P particle comprising a norovirus epitope and/or motif selected from the group consisting of:
a) Epitope A;
b) Epitope B;
c) Epitope C;
d) Epitope D;
e) Epitope E;
f) NEPvK motif; and
g) any combination of (a) through (f) above,
wherein the epitope and/or motif is presented on the P particle surface,
6. A mimitope comprising a norovirus epitope and/or motif selected from the group consisting of:
a) Epitope A;
b) Epitope B;
c) Epitope C;
d) Epitope D;
e) Epitope E;
f) NERK motif; and
g) any combination of (a) through (f) above.
7. The chimeric norovirus capsid protein of claim 1, the synthetic backbone molecule of claim 4, the norovirus P particle of claim 5 or the mimitope of claim 6, comprising Epitope A from GII.4.1974, GII.4.1987, GII.4.1997, GIIA2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope A is from a norovirus strain different from the norovirus VP1 major capsid protein backbone.
8. The chimeric norovims capsid protein of claim 1, the synthetic backbone molecule of claim 4, the norovims P particle of claim 5 or the mimitope of claim 6, comprising Epitope B from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope B is from a norovims strain different from the norovirus VP1 major capsid protein backbone.
9. The chimeric norovirus capsid protein of claim 1, the synthetic backbone molecule of claim 4, the norovirus P particle of claim 5 or the mimitope of claim 6, comprising Epitope C from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GIL4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope C is from a norovirus strain different from the norovirus VP1 major capsid protein backbone.
10. The chimeric norovirus capsid protein of claim 1 , the synthetic backbone molecule of claim 4, the norovirus P particle of claim 5 or the mimitope of claim 6, comprising Epitope D from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s, GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope D is from a norovims strain different from the norovims VP1 major capsid protein backbone.
1 1. The chimeric norovirus capsid protein of claim 1 , the synthetic backbone molecule of claim 4, the norovims P particle of claim 5 or the mimitope of claim 6, comprising Epitope E from GII.4.1974, GII.4.1987, GII.4.1997, GII.4.2002a, GII.4.2002, GII.4.2004, GII.4.2005, GII.4.2006, GII.4.2007, GII.4.2008s? GII.4.2008a, GII.4.2009 or GII.4.2012, wherein Epitope E is from a norovirus strain different from the norovims VP1 major capsid protein backbone
12. The chimeric norovims capsid protein of claim 2, wherein the norovirus VP1 major capsid protein backbone is from GII.4.2006.
13. The chimeric norovims capsid protein of claim 12, comprising Epitope A from GII.4.1987 and Epitope E from GII.4.2002.
14. An isolated nucleic acid molecule encoding the chimeric norovims capsid protein of any of claims 1-3 and 7-13.
15. A vector comprising the nucleic acid molecule of claim 14.
16. A Venezuelan Equine Encephalitis (VEE) replicon particle (VRP) comprising the nucleic acid molecule of claim 14.
17. A virus like particle (VLP) comprising the chimeric noro virus capsid protein of any of claims 1-3 and 7-13.
18. A composition comprising the chimeric noro virus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the nucleic acid molecule of claim 14, the vector of claim 15, the VRP of claim 16 and/or the VLP of claim 17 in a pharmaceutically acceptable carrier.
19. A method of producing an immune response to a norovirus in a subject, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the nucleic acid molecule of claim 14, the vector of claim 15, the VRP of claim 16, the VLP of claim 17 and/or the composition of claim 18.
20. A method of treating a norovirus infection in a subject in need thereof, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the nucleic acid molecule of claim 14, the vector of claim 15, the VRP of claim 16, the VLP of claim 17 and/or the composition of claim 18.
21. A method of preventing a disorder associated with norovirus infection in a subject, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of any of claims 1-3 and 7-1 1 , the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the nucleic acid molecule of claim 14, the vector of claim 15, the VRP of claim 16, the VLP of claim 17 and or the composition of claim 18.
22. A method of protecting a subject from the effects of norovirus infection, comprising administering to the subject an effective amount of the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the raimitope of claim 6, the nucleic acid molecule of claim 14, the vector of claim 15, the VRP of claim 16, the VLP of claim 17 and/or the composition of claim 18.
23. A method of treating a noro virus infection in a subject, comprising administering to the subject an effective amount of a humanized GII.4.2002.G5 antibody,
24. A method of protecting a subject from the effects of noroviras infection, comprising administering to the subject an effective amount of a humanized GII.4.2002.G5 antibody.
25. A method of detecting a neutralizing antibody to a norovirus, the method comprising determining whether an antibody binds to the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17, wherein binding by the antibody to the chimeric norovirus capsid protein of any of claims 1 -3 and 7- 11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17 indicates that the antibody is a neutralizing antibody to a norovirus.
26. A method of identifying a neutralizing antibody to a norovirus, comprising:
(a) contacting an antibody with the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17; and
(b) determining if the antibody binds to the chimeric norovirus capsid protein of any of claims 1-3 and 7-1 1, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17, wherein binding by the antibody to the chimeric norovirus capsid protein of any of claims 1 -3 and 7-1 1, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17 identifies the antibody as a neutralizing antibody to a norovirus.
27. A method of identifying an immunogenic composition that induces a neutralizing antibody to a norovirus in a subject, the method comprising: (a) contacting a biological sample from a subject that has been administered the immunogenic composition with the chimeric norovirus capsid protein of any of claims 1 -3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17;
(b) determining if the biological sample comprises an antibody that binds the chimeric norovirus capsid protein of any of claims 1-3 and 7-1 1, the synthetic backbone molecule of claim 4S the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17; and
(c) identifying the immunogenic composition as inducing a neutralizing antibody to a norovirus in the subject if the biological sample comprises an antibody that binds to the chimeric norovirus capsid protein of any of claims 1-3 and 7-11 , the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17.
28. A method of identifying an immunogenic composition that induces a neutralizing antibody to a norovirus in a subject, the method comprising:
(a) administering an immunogenic composition comprising a norovirus antigen to a subject in an amount effective to induce antibodies against the norovirus antigen;
(b) contacting a biological sample from the subject with the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17;
(c) determining if the biological sample comprises an antibody that binds the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17; and
(d) identifying the immunogenic composition as inducing a neutralizing antibody to a norovirus in the subject if the biological sample comprises an antibody that binds the chimeric norovirus capsid protein of any of claims 1-3 and 7-11, the synthetic backbone molecule of claim 4, the P particle of claim 5, the mimitope of claim 6, the VRP of claim 16 and/or the VLP of claim 17.
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