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WO2017011394A1 - Anticorps monoclonaux humains dirigés contre le norovirus humain et découverte d'épitopes - Google Patents

Anticorps monoclonaux humains dirigés contre le norovirus humain et découverte d'épitopes Download PDF

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Publication number
WO2017011394A1
WO2017011394A1 PCT/US2016/041763 US2016041763W WO2017011394A1 WO 2017011394 A1 WO2017011394 A1 WO 2017011394A1 US 2016041763 W US2016041763 W US 2016041763W WO 2017011394 A1 WO2017011394 A1 WO 2017011394A1
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Prior art keywords
seq
antibody
binding
norovirus
antibodies
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Inventor
Mary K. Estes
Robert Legare Atmar
B V Venkatar PRASAD
James Crowe
Gopal SAPPARAPU
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Baylor College of Medicine
Vanderbilt University
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Baylor College of Medicine
Vanderbilt University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Noroviruses are important human pathogens (FIG. 1). They are icosahedral viruses made up of 90 dimers of the major capsid protein VP1. The subunit structure is comprised of two principal domains: the S domain forms the shell and the P domain that projects out from the shell. The P domain is further divided into two subdomains, PI and P2. The distally located P2 subdomain can be considered as a large insertion into the PI domain.
  • HBGA histo-blood group antigens
  • These glycoconjugates are also susceptibility factors for these viruses. These HBGAs bind to the P2 subdomain.
  • the P2 subdomain is the least conserved region of the norovirus capsid protein leading to significant variations in the HBGA specificity and antigenicity.
  • the interplay between variations in antigenicity and HBGA specificity are suggested to drive the norovirus evolution.
  • Recent studies have shown that circulating serum antibodies that block HBGA binding correlate with protection.
  • human noroviruses are classified into several genogroups and genotypes (FIG. 2).
  • the HBGA binding site for several of these genogroups has been structurally well characterized. However, the structural basis of how 'neutralizing' antibodies block HBGA binding is not known.
  • Embodiments of the disclosure concern monoclonal antibodies to human norovirus and methods of their use.
  • Monoclonal antibodies described herein can be used at least to analyze samples from animals, including biological fluids from individuals with or suspected of having Norovirus infection.
  • the antibody can be a mouse antibody, a human antibody, or a humanized antibody, for example.
  • the antibody is a mouse monoclonal antibody.
  • the antibody is a human or humanized monoclonal antibody.
  • the antibody is formulated in a pharmaceutically acceptable formulation.
  • compositions encompassed by the disclosure can be used as a novel treatment for Norovirus infection.
  • antibodies of the disclosure can be used to reduce or eliminate Norovirus infection.
  • the disclosure provides methods for treating Norovirus infection or related sequelae comprising the step of administering an effective amount of an antibody (or multiple antibodies) encompassed by the disclosure or a peptide comprising or consisting of the amino acid sequence for a conformational epitope formed by surface-exposed loop clusters in the P domain in the capsid protein, including some or all of the residues N346, T348, D350, F352, S380, H381, S383, N394, and G396.
  • conformational antibody that specifically binds the surface-exposed loop cluster of the P domain of the capsid protein.
  • the conformational epitope includes one or more amino acid residues selected from N346, T348, D350, F352, S380, H381, S383, N394, and G396 relative to SEQ ID NO: 61.
  • the conformational antibody specifically binds an epitope comprising amino acid residues N346, T348, D350, F352, S380, H381, S383, N394, and G396 relative to SEQ ID NO: 61.
  • Embodiments of the disclosure concern the structural and mechanistic basis of HBGA binding with Noroviruses (NoVs).
  • Embodiments of the disclosure provide the first crystal structure of the Norwalk virus P domain in complex with the FAB fragment of an HBGA blockade antibody, IgA 512.
  • sequence differences and structural alterations in other genotypes play a role in the ability of other gentoypes to escape from IgA 512 neutralization.
  • Described herein is a set of monoclonal antibodies made to human norovirus that are useful for therapy, prevention, diagnostic tests, and/or characterization of viral vaccines or VLPs prior to release.
  • the disclosure provides the amino acid sequence(s) of an epitope recognized by inhibitory antibodies, for example that can be used in designing new vaccines.
  • the disclosure provides a structural description of how an antibody binds to human norovirus and blocks binding of histo-blood group antigens (HBGAs), which are the initial receptors for these viruses.
  • HBGA blocking by antibodies is a mechanism that can be used to neutralize the virus.
  • the monoclonal antibody is a human monoclonal antibody.
  • Certain embodiments are directed to methods of treating or preventing Norovirus infection in individuals in need thereof, including those susceptible to the infection or at risk for the infection.
  • the individual may be at risk for the infection by having an impaired immune system or being exposed to large numbers of individuals, for example in a confined environment, such as in a school, transportation vessel (boat, plane, train), sports or entertainment venue, etc.
  • the compositions may be provided to an individual as a precautionary measure or as a routine measure. Any individual of any age or gender may be exposed to methods and/or compositions of the disclosure.
  • antibodies and binding polypeptides comprising amino acid sequences that comprise or consist of or consist essentially of the amino acid sequences of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:40, SEQ ID NO:40
  • a functionally active derivative thereof is a polypeptide that can bind a Norovirus and block its binding to histoblood group antigens (glycans) and have a 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO
  • Any individual suspected of having or known to have Norovirus infection or exposure or at risk thereof may be provided a therapeutically effective amount of one of the aforementioned antibodies or binding polypeptides.
  • the polypeptides or antibodies may be delivered in any suitable manner, e.g., liposomes.
  • nucleic acid sequences that comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and functional derivatives thereof that are at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84
  • nucleic acids may be delivered in any manner, including in a vector or in the absence of a vector.
  • vectors include liposomes, viral vectors, non-viral vectors, and so forth.
  • non-viral vectors include plasmids.
  • viral vectors include adeno-associated virus, adenoviral virus, vaccinia virus, retroviral virus, lentivirus, and so forth.
  • the nucleic acid may be delivered as RNA, also.
  • compositions for therapeutic purpose may occur by any suitable regimen, including being administered more than one time to the subject, and they may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more times.
  • the route of administration of the compositions includes, but is not limited to oral, parenteral, subcutaneous and intravenous administration, rectal, or various combinations thereof, including inhalation or aspiration.
  • Certain embodiments are directed to diagnostic tests employing antibodies or binding polypeptides of the present disclosure.
  • the diagnostic tests may include the HBGA- blocking monoclonal antibodies encompassed herein and may test whether or not the individual is infected with Norovirus.
  • the diagnostic tests may include more than one type of monoclonal antibody, for example in situations where an individual is being tested for more than one genotype of Norovirus. Any suitable sample from the individual may be employed, including at least stool and/or vomitus.
  • a monoclonal antibody is a human monoclonal antibody.
  • a peptide comprising surface-exposed loop clusters in the P domain in the capsid protein (and in some cases that can form a conformational epitope formed by surface-exposed loop clusters in the P domain from the capsid protein, including some or all of the residues N346, T348, D350, F352, S380, H381, S383, N394, and G396) can be used to induce antibodies that reduce Norovirus infection or detect Norovirus infection.
  • a peptide comprising the epitope comprising amino acid residues 344, 346, 348, 351, 381, 383, and 385 of GenBank® Accession No. AAB50466 (SEQ ID NO: 61).
  • the antibody binds a conformation- specific or conformational epitope or recognizes a conformational epitope located in a surface-exposed loop in the P2 subdomain.
  • FIG. 1 General information about Noroviruses.
  • FIG. 4. Demonstration that the monoclonal antibodies are specific to GI.l and exhibit HBGA blockade.
  • FIG. 5 Structure of Noro virus P domain complexed with human IgA 512.
  • FIG. 6 Illustration that IgA 512 binds to P domain through CDRs H3, LI and
  • FIG. 7 Illustration of a close-up of Pdomain-512 interactions.
  • FIG. 8 Structure of Norovirus P domain bound to H type HBGA.
  • FIG. 10 Sequence and structure and alterations mediate IgA 512 genotype specificity.
  • FIG. 11 IMGT analysis of particular antibodies (SEQ ID NOs. 90-108).
  • FIG. 12 Fab 512 binds tightly to NV P domain.
  • P domain- Fab 512 association- dissociation curves were obtained through serial two fold dilutions of Fab 512 (0.5-0.015 ⁇ ) plus buffer controls using the Octet acquisition software. Sensograms for all concentrations are shown and labeled accordingly. The calculated KD, Kon, and Koff are shown in a tabular form.
  • FIG. 13 Fab 512 recognizes a conformational epitope on top of NV P domain.
  • Cartoon representation of the overall structure of Fab 5I2-P domain complex showing one Fab 512 molecule bound to each subunit of the P domain dimer. The individual P dimer subunits are shown in blue and green with PI and P2 subdomains labeled. The dashed black line indicates the two fold symmetry axis.
  • Fab 512 is depicted with the heavy and light chains shown in cyan and magenta, respectively. The variable and constant domains of Fab 512 light chain and heavy chain are labeled VL-CL and VH- CH respectively. The N and C terminal are also indicated for both P domain and Fab 512.
  • FIG. 14A Close up view of Fab 512 bound to P domain (blue) bound to Fab 512 (heavy chain: cyan, light chain: magenta) depicted in both surface and cartoon representation. All the six CDRs, three from the light chain (L1-L3) and three from the heavy chain (H1-H3), were identified and labeled respectively. Similarly six loop regions were identified on the P2 subdomain. Five of these loops have been previously identified in other genotypes and labeled according to convention ( loops- A, B, P, T and U). The sixth loop labeled loop Q was identified in this disclosure.
  • FIG. 14B Molecular details of Fab 5I2-P domain interactions.
  • Fab 512 binds P domain through CDRs LI, L2 and H3 that make a network of hydrogen bonding (black dashed lines) and hydrophobic interactions (red dotted line) with the loops U, Q and T of the P domain.
  • CDRLl makes the predominant interactions. All interacting loops are shown in cartoon representation with interacting residues shown as stick model as per above color convention with nitrogen and oxygen atoms in blue and red respectively.
  • FIGS. 15 A, 15B, 15C, and 15D Complementary surface residues involved in Fab 5I2-P domain interaction. Presentation of complementary surfaces is important to antigen- antibody recognition and binding; two pockets, one on the P domain surface (FIG. 15A) and one on the Fab surface (FIG. 15C), were identified in the disclosure and are shown to accommodate a complementary residue from the other molecule.
  • FIG. 15A A pocket on the P domain surface (blue) buries a lysine 32 residue (pink stick model) contributed by the CDRLl of Fab 512.
  • FIG. 15C A similar pocket on the surface of Fab 512 (magenta) is shown to accommodate a histidine residue (H381) (yellow stick model). H381 makes hydrophobic and stacking interactions with three tyrosine residues labeled Y31, Y38 and Y98 contributed by CDRs LI and L3 of Fab 512.
  • FIGS. 15B and 15D Interestingly, superposition of the Fab 512 bound P domain structure and native NV VLP structure (PDB ID.
  • FIG. 15B shows that Fab binding induces local conformational changes on P domain to make favorable interactions.
  • FIG. 15B The loop U moves about 10A to make favorable interactions with CDRLl and forms one side of the pocket that buries residue K32. Loop U from VLP is labeled in grey and marked with an asterix the movement of loop U is indicated by an arrow.
  • FIG. 15D Similarly, Fab binding induces a flip in the orientation of the side chain of a H381, allowing it to make favorable hydrophobic and stacking interactions. In the native VLP structure the sidechain of H381 (grey, indicated with asterisk) would sterically clash with Y98 residue of CDRL3.
  • FIGS. 16A, 16B, 16C, and 16D Fab 512 blocks HBGA binding to P domain through steric hindrance.
  • Superposition of HBGA bound P domain (PDB ID. 2ZL6) and the Fab 512 bound P domain structure reveals steric hindrance as the mechanism of HBGA blockade.
  • FIG. 16A Surface representation (grey) and cartoon representation of the P domain dimer (side view) bound to H type HBGA (yellow sticks).
  • FIG. 16B Superposition of Fab and HBGA bound P domain structures clearly shows that Fab 512 will sterically hinder binding of HBGA.
  • FIGS. 17A and 17B Sequence and structural changes mediate escape from Fab 512 neutralization in other genotypes.
  • FIG. 17A Amino acid sequence alignment of representative GI variants showing the poor conservation of residues at positions that correspond to residues from the three loop regions in GI.l that are involved in interacting with Fab 512. The residues in loop Q, T and U are colored in red, yellow and green respectively.
  • FIG. 17B The residues in loop Q, T and U are colored in red, yellow and green respectively.
  • FIGS. 18A and 18B Mapping of neutralizing epitopes on NoVs.
  • Cluster 1 (red) comprises the evolving residues in the T loop.
  • Cluster 2 (blue) comprises residues in the Q and U loops.
  • Cluster 3 (green) comprises residues in the A and B loops.
  • the identified clusters are in close proximity to the HBGA binding site (yellow). NAb's can either bind to individual clusters or use a combination of these clusters to bind and neutralize NoVs.
  • Epitope of Fab 512 is located in clusters land 2 and is indicated by a dotted line and labeled respectively.
  • FIGS. 19A and 19B Screening supernatants of EBV-transformed B cell cultures from two NoV-challenged subjects.
  • B cell culture supernatants were added to replicate microtiter plates coated with NoV VLP and probed with a mixture of (i) a mixture of anti-human ( ⁇ + ⁇ ; to determine the total number of binders), or (ii) anti-human IgG ( ⁇ - specific; to determine IgG frequency), or (iii) anti human IgA (a-specific; to determine the IgA frequency) secondary antibodies.
  • Blocking assay was done as described in Methods.
  • the number of binding (A450 >1.5) and blocking (A450 ⁇ 2.1) were counted and percent distribution among binders and blockers was calculated. Distribution of IgG (red) or IgA (blue) classes of antibodies that bound to NoV VLP (FIG. 19A) or blocked VLP - glycan interaction (FIG. 19B) is shown.
  • FIGS. 20A and 20B Binding and blocking characteristics of purified monoclonal antibodies.
  • Purified IgG (red) or IgA (blue) antibodies were tested for binding to NV VLP in ELISA (FIG. 20A) or for blocking VLP - glycan interaction (FIG. 20B).
  • Each of the IgG antibodies bound to VLPs with lower EC50 values than IgA antibodies, while in contrast the concentrations needed for blocking were similar for IgG and IgA.
  • the blocking of murine mAb 8812 is shown in black.
  • FIGS. 21A, 21B, and 21C Specificity of human mAbs.
  • the binding (mean absorbance at 450 nm + SD) of purified mAbs at 20 ⁇ g/mL to VLPs representing homologous virus (NoV GI.I) or heterologous human NoVs of different genotypes (FIG. 21A) or antigens representing wild-type or mutant recombinant capsid proteins of homologous virus (FIG. 21B) were assessed by ELISA to evaluate genotype specificity and to infer the subdomain of major capsid protein bound by anti norovirus mAbs.
  • the data shown in each figure summarizes the results from 2 independent experiments. (FIG.
  • FIGS. 22A, 22B, and 22C Nature of epitopes recognized by anti-norovirus mAbs.
  • Norovirus VLPs were resolved on SDS-PAGE gels under (FIG. 22A) nonreducing, nondenaturing, or (FIG. 22B) reducing, denaturing conditions and the membranes were probed with anti-norovirus mAbs. All the human antibodies, and the murine mAb 8812, bound to conformational epitopes, while denatured VLP were bound only by mAb 3901. Arrowhead in panel B indicates VP1.
  • FIG. 22C Antibodies were binned into competition- binding groups in ELISA as described in Methods. Most of the antibodies seem to compete for the same or spatially proximate epitopes. The asymmetric nature of competition suggests subtle factors such as the angle of approach of the antibodies seem to have an effect on competition.
  • FIGS. 23 A and 23B Molecular assembly of hybridoma-derived antibodies obtained from Donor 1.
  • IgA (blue) and IgG (red) antibodies were purified by affinity chromatography and resolved on SDS polyacrylamide gels under reducing, denaturing conditions (FIG. 23A) or non-reducing conditions (FIG. 23 B) and stained with Coomassie Blue. Monomeric (*) and dimeric (**) forms of IgA are evident.
  • FIGS. 24A and 24B Average binding and blocking profiles of IgG and IgA antibodies.
  • the dose-response curves for binding (FIG. 24A) or blocking (FIG. 24B) for all IgG and IgA antibodies were averaged to generate representative curves for each class using R software package.
  • FIG. 25 Variable heavy and light chain domains of anti-NoV mAbs were cloned into expression vectors containing ⁇ or a constant domains for heavy chains, and ⁇ or ⁇ constant domains for the light chains. Antibodies were expressed transiently in HEK293 cells. For expression of dig A, a plasmid coding for J chain was cotransfected with the heavy and light chains. Antibodies purified from supernatant by affinity chromatography were resolved on SDS-PAGE gels under nonreducing conditions and stained with Coomassie Blue reagent.
  • FIG. 26 Representative curves for blocking assays with isotype switch variants for each of the antibody clones. IgG or monomeric (mlgA) or dimeric (dig A) forms of IgA were used in the HBGA blocking assay. Results are shown with concentration of Ab as loglO nM combining sites). [0042] FIG. 27. Genetic characteristics of anti-norovirus mAbs (SEQ ID NOs. 109-
  • isolated can refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized).
  • an isolated compound refers to one that can be administered to a subject as an isolated compound; in other words, the compound may not simply be considered “isolated” if it is adhered to a column or embedded in an agarose gel.
  • an "isolated nucleic acid fragment” or “isolated peptide” is a nucleic acid or protein fragment that is not naturally occurring as a fragment and/or is not typically in the functional state.
  • Embodiments of the disclosure include molecules of any type that target a particular epitope to prevent binding thereto.
  • the molecules that target the epitope are peptide(s) or polypeptide(s), including antibodies or fragments thereof.
  • the targeting of a particular epitope includes direct binding to the epitope.
  • the epitope comprises part or all of the histo-blood group antigens (HBGA) binding site on the P domain of VP1 of human Norovirus.
  • HBGA histo-blood group antigens
  • Specific aspects provide for an antibody that binds to a site on Norovirus that sterically blocks HBGA from accessing the binding site.
  • the antibody is an IgA antibody, such as to facilitate mucosal immunity.
  • the epitope is a conformational epitope formed by two surface-exposed loop clusters in the P domain of VP1 of human Norovirus.
  • the epitope comprises H381 of GI. l P domain and includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues on one or both sides of H381 of the polypeptide.
  • the epitope may or may not have contiguous amino acids from a particular sequence involved.
  • the amino acid sequence of an epitope of the disclosure comprises residues in the P domain in the capsid protein, including some or all of the residues N346, T348, D350, F352, S380, H381, S383, N394, and G396.
  • Embodiments of the disclosure include antibodies or antibody fragments that recognize the epitope that comprises residues in the P domain in the capsid protein, including some or all of the residues N346, T348, D350, F352, S380, H381, S383, N394, and G396.
  • the antibody may be polyclonal or monoclonal.
  • a monoclonal antibody or antibody fragment specifically binds an amino acid sequence comprising residues in the P domain in the capsid protein, including some or all of the residues N346, T348, D350, F352, S380, H381, S383, N394, and G396.
  • the monoclonal antibody or antibody fragment may be a human monoclonal antibody, human monoclonal antibody fragment, a mouse monoclonal antibody, or a mouse antibody fragment. In some cases, the monoclonal antibody is a single chain antibody. The monoclonal antibody or antibody fragment may be a humanized monoclonal antibody or antibody fragment. In certain other embodiments, the antibody is a human antibody. In still further aspects the antibody is a recombinant antibody segment. An antibody may be isolated, chimeric, non-natural, and/or recombinant.
  • antibody binding occurs through one or more of complementarity determining loop (CDRL) 1 in light chain, CDRL3 in light chain, and complementarity determining region (CDR) loop (CDRH3) in heavy chain.
  • CDRL complementarity determining loop
  • CDRH3 complementarity determining region
  • the antibody binds the P2 subdomain through the CDRL1 and the histidine residue H381.
  • any polypeptide of the disclosure targets a Norovirus of the genotype GI.1.
  • Embodiments of the disclosure include binding polypeptides that bind Norovirus particle P domain through one or more of complementarity determining loop 1 in the antibody light chain (CDRL1), CDRL3 in the light chain, and complementarity determining region (CDR) 3 loop in the antibody heavy chain (CDRH3).
  • CDRL1 complementarity determining loop 1 in the antibody light chain
  • CDRL3 complementarity determining region
  • CDRH3 complementarity determining region 3 loop in the antibody heavy chain
  • Embodiments of the disclosure include compositions that comprise the antibody, and in some cases the composition(s) comprise a monoclonal antibody or a mixture of two or more different monoclonal antibodies.
  • the different antibodies may or may not target the same epitope or protein or genotype of Norovirus.
  • small molecule mimics that specifically target the HBGA binding site are utilized.
  • any composition encompassed by the disclosure is in a pharmaceutically acceptable formulation.
  • the antibodies described herein may block HBGA binding.
  • the antibodies encompassed by the disclosure may block HBGA through direct competition for the HBGA binding site, through allosteric disruption of the HBGA binding site by inducing conformational changes in the P domain, or by steric masking of the HBGA binding site, or another mechanism.
  • an antibody of the disclosure blocks HBGA predominantly by steric hindrance.
  • antibodies with a prevalent involvement of CDRL1 in antigen recognition are employed.
  • an antibody is utilized that comprises a length of the CDR of CDRL1 that is longer than typical lengths, including loop lengths longer than 11, 12, 13, 14, 15, 16, or 17 amino acids, for example.
  • an antibody is utilized that has a length of CDRLl that is longer than typical (in k chain human antibodies, the length of the CDRLl varies between 10 and 17 residues, with the majority of the antibodies exhibiting a loop length of 11 residues) and in combination also has an H3 loop with fewer than typical amino acids involved in antigen specificity (in IgA 512, for example, the H3 loop is positioned slightly away from the P2 subdomain, with just two of its residues interacting with the P2 subdomain.
  • CDRH3 encoded by the highly diverse D-JH joining genes plays a dominant role because of the inherent sequence diversity and consequent conformational variability).
  • Embodiments of the disclosure include a crystal structure of a HBGA- blocking monoclonal antibody bound to Norovirus, revealing, in one embodiment, its mechanism of neutralization.
  • Certain embodiments are directed to a hybridoma cell and to a monoclonal antibody produced by a hybridoma.
  • Certain aspects include particular monoclonal antibodies, and sequences thereof. Sequences of the particular monoclonal antibody 512 are as follows (CDRs are underlined):
  • Monoclonal antibodies other than 512 are encompassed in the disclosure.
  • a monoclonal antibody is encoded by a particular nucleotide sequence.
  • Nucleotide sequences of HBGA-blocking monoclonal antibodies other than 512 are as follows:
  • NV2J3 light CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCG ATCACCATCTCCTGCACTGGAACCATCAGTGATGTTGGTGGTTATAACTATGTCT CCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCA ATAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACAC GGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTAATTATTACTGC TGCTCATATGCAACTAGTACCAATTTGCTATTCGGCGGAGGGACCCAGCTGACCG TCCTA (SEQ ID NO: l l)
  • NV2L8 HEAVY EFQLVQSGAEVKKPGASVKVSCKASGYTFRKYYMHWVRQAPGQGPEWMGI INPSGGNTGYAQKFQGRVTVTRDTSTSTVYMELSSLRSEDTAVYYCARGGISWYVT GFD YWGQGTLVT VS S AS F (SEQ ID NO:31)
  • a monoclonal antibody that comprises one or more sequences selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.
  • Embodiments are directed to monoclonal antibody polypeptides, polypeptides having one or more segments thereof, and polynucleotides encoding the same.
  • a polypeptide can comprise all or part of the heavy chain variable region and/or the light chain variable region of Norovirus- specific antibodies.
  • a polypeptide can comprise an amino acid sequence that corresponds to a first, second, and/or third complementary determining regions (CDRs) from the light variable chain and/or heavy variable chain of an antibody, e.g. , a Norovirus-specific antibody.
  • CDRs complementary determining regions
  • an antibody or binding polypeptide may have a binding region comprising an amino acid sequence having, having at least, or having at most 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
  • an antibody having all or part of one or more CDRs disclosed herein has been humanized in non-CDR regions.
  • the CDR regions disclosed herein may be changed by 1 ,2 ,3 ,4, 5, 6, 7 or 8 amino acids per CDR, which may be instead of or in addition to humanization.
  • a change may be a deletion or addition of 1, 2, or 3 amino acids, or it may be a substitution of any amino acid, which may or may not be with an amino acid that is a conserved an amino acid.
  • a Norovirus -binding polypeptide or antibody has one, two, three, four, five, or six CDRs that have or have at least 40, 45, 50, 55, 60, 65, 70, 75, 80,
  • a Norovirus -binding polypeptide or antibody has an amino acid sequence corresponding to CDRl, CDR2, and CDR3 of a light chain variable region and a CDRl, CDR2, and CDR3 of a heavy chain variable region.
  • the amino acid sequence corresponding to a CDR may have a percent identity to a CDR encompassed herein.
  • a polypeptides described herein comprise one or more amino acid segments of any of the amino acid sequences disclosed herein.
  • a polypeptide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid segments comprising about, at least or at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
  • amino segment(s) are selected from one of the amino acid sequences provided herein.
  • embodiments of the disclosure include an antigen comprising the sequence of a peptide designed to mimic the P domain in the capsid protein, including some or all of the residues N346, T348, D350, F352, S380, H381, S383, N394, and G396.
  • a nucleic acid molecule of the embodiments comprises one or more nucleic acid segments of the any of the nucleic acid sequences disclosed herein.
  • a nucleic acid molecule can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid segments comprising about, at least or at most 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
  • compositions comprising one or more polypeptides or antibodies or antibody fragments that are encompassed herein.
  • Such a composition may or may not contain additional active ingredients.
  • compositions comprising, consisting of, or consisting essentially of a polypeptide comprising one or more antibody fragments encompassed herein. It is contemplated that the composition may contain non- active ingredients.
  • the antibody may comprise one or more detectable agents, such as a radioactive marker, a nucleic acid, a fluorescent label, or an enzymatic label, and so forth.
  • compositions or antibodies described herein may be utilized in the treatment, prevention, and/or detection of Norovirus infection for a mammal, including at least a human, dog, cat, horse, pig, sheep, goat, and so forth, and/or an environment.
  • the compositions or antibodies are useful for the treatment, prevention, and/or detection of any genotype of Norovirus, although in some cases the compositions or antibodies are useful for the treatment, prevention, and/or diagnosis of a particular genotype or subcombination of genotypes.
  • the compositions or antibodies are useful for Norovirus genotype GI.l, for example.
  • compositions or antibodies, or mixtures thereof are delivered prior to and/or following exposure of an individual to large populations of individuals or environments prone to Norovirus infection, including confined environments.
  • Such environments include passenger vessels, including cruise ships, airplanes, and trains; schools; arenas; military environments, such as military encampments; health care facilities, including nursing homes, hospitals, and long-term care facilities; food service settings, such as restaurants and catered events; child care centers; prisons; recreational water settings; and so forth.
  • the compositions or antibodies may be additionally or alternatively provided to an individual in the course of routine preventative measures.
  • an individual is provided antibody composition(s) for the prevention of Norovirus infection.
  • individual antibodies are effective for one genotype, and therefore an individual is given a plurality of antibodies, each specific for a Norovirus genotype.
  • an individual is given the antibody composition(s) in multiple administrations, such as through booster deliveries.
  • the antibodies encompassed herein can be used in immunohistochemical and biochemical methods for qualitative and/or quantitative analysis of samples from an individual suspected of having Norovirus infection.
  • Further aspects are directed to methods for evaluation of an individual suspected of having Norovirus.
  • the method of evaluating an individual suspected of or having Norovirus comprises the step of detecting binding of an antibody that specifically binds to a particular Norovirus epitope in a biological sample from the individual, wherein the detection in the biological sample is indicative of the presence of Norovirus.
  • the detection may be by immunoassay, for example.
  • a biological sample from an individual for analysis in methods of the disclosure may comprise stool, vomitus, saliva, serum, plasma, or tissue specimens for histopathology such as intestinal biopsy specimens.
  • food for example, shellfish, including mollusks such as oysters, clams, mussels and scallops
  • water and/or environment samples (including swabs of environmental surfaces) are tested for the presence of Norovirus using antibodies encompassed by the disclosure.
  • compositions include Norovirus-binding polypeptides in amounts effective to achieve the intended purpose - treatment or protection of Norovirus infection.
  • binding polypeptide refers to a polypeptide that specifically binds to a target molecule, such as the binding of an antibody to an antigen. Binding polypeptides may but need not be derived from immunoglobulin genes or fragments of immunoglobulin genes. More specifically, an effective amount means an amount of active ingredients necessary to provide resistance to, amelioration of, or mitigation of infection.
  • an effective amount prevents, alleviates or ameliorates symptoms of disease or infection, or prolongs the survival of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • an effective amount or dose can be estimated initially from in vitro, cell culture, and/or animal model assays. For example, a dose can be formulated in animal models to achieve a desired response. Such information can be used to more accurately determine useful doses in humans.
  • Any of the compositions and methods of using these compositions can treat a subject having, suspected of having, or at risk of developing a Norovirus infection or related disease. One use of the compositions is to prevent infections by inoculating a subject prior to exposure to Norovirus.
  • a method for treating a Norovirus infection comprising the step of administering an effective amount of antibody that specifically binds an epitope comprising a conformational epitope formed by surface-exposed loop clusters in the P domain in the capsid protein, including some or all of the residues N346, T348, D350, F352, S380, H381, S383, N394, and G396 to an individual having or suspected of having a Norovirus infection.
  • an individual may also be treated for dehydration, including oral rehydration fluids and/or intravenous fluids.
  • the dose of antibody delivered to the individual a dose of 0.1, 0.5, 1, 5, 10, 50, 100 mg or g/kg to 5, 10, 50, 100, 500 mg or g/kg.
  • the antibody is delivered to the individual via a route that is intravenous, intramuscular, and/or oral.
  • Certain aspects are directed to methods of preventing or treating Norovirus infection comprising administering to an individual having or suspected of having a Norovirus infection an effective amount of one or more purified polypeptides or proteins that specifically bind the P2 subdomain of the Norovirus.
  • the polypeptides bind a conformational epiropte that includes the H381 residue of the P2 subdomain of the protein sequence of GenBank® Accession No. AAB50466 or a corresponding residue(s) define corresponding. It is contemplated that this polypeptide (or protein) may be referred to as an antibody by virtue of it being a polypeptide or protein with amino acid sequences of or derived from one or more CDR regions of an antibody.
  • any embodiment discussed herein in the context of an antibody may be implemented with respect to a polypeptide or protein so long as the polypeptide or protein has one or more amino acid regions that has at least 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to a CDR from an antibody that is capable of specifically binding the P2 subdomain.
  • the binding polypeptide can be a purified polyclonal antibody, a purified monoclonal antibody, a recombinant polypeptide, or a fragment thereof.
  • the polypeptide is an antibody that is humanized, which means the non- variable portion of the antibody has been altered in order to simulate the constant regions found in human antibodies.
  • a humanized antibody is one that has the CDR sequences of a non-human antibody (or at least amino acid sequences that are derived from such sequences, i.e., are at least 70%, 75%, 80%, 85%, 90%, 95%, or greater in identity).
  • Certain aspects are directed to methods of treating a subject having or suspected of having a Norovirus infection comprising administering to a patient having or suspected of having a Norovirus infection an effective amount of a purified antibody or binding polypeptide that specifically binds Norovirus, including the P2 subdomain, including at or near H381 residue or a residue as noted in FIG. 10.
  • the antibody or binding peptide binds a conformation epitope that includes the H381 residue of GenBank® Accession No. AAB50466 (SEQ ID NO:61) or a residue as noted in FIG. 10.
  • methods are directed to treating a subject at risk of a Norovirus infection comprising administering to a patient at risk of a Norovirus infection an effective amount of an antibody or binding polypeptide that binds a Norovirus P2 polypeptide prior to infection with Norovirus.
  • compositions include antibodies in amounts effective to achieve the intended purpose - treatment or protection of Norovirus infection. More specifically, an effective amount means an amount of active ingredients necessary to provide resistance to, amelioration of, or mitigation of infection. In more specific aspects, an effective amount prevents, alleviates or ameliorates symptoms of disease or infection, or prolongs the survival of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • an effective amount or dose can be estimated initially from in vitro, cell culture, and/or animal model assays.
  • a dose can be formulated in animal models to achieve a desired response. Such information can be used to more accurately determine useful doses in humans.
  • compositions and methods of using these compositions can treat a subject having, suspected of having, or at risk of developing a Norovirus infection or related disease.
  • One use of the compositions is to prevent infections by inoculating a subject prior to exposure to Norovirus.
  • a method of treating an individual for Norovirus infection or preventing Norovirus infection in an individual comprising the step of providing to the individual a therapeutically effective amount of a binding polypeptide or an antibody that blocks the binding of the virus to the HBGA.
  • the binding polypeptide or an antibody binds the exposed loops in the P2 subdomain of the Norovirus particle. In a further aspect the binding polypeptide or an antibody binds an epitope comprising residue H381 of a Norovirus particle. In still a further aspect the binding polypeptide or antibody binds through one or more of complementarity determining loops in the light chain (e.g., CDRL1 or CDRL3); and CDRH3 in the heavy chain. In certain aspects the binding polypeptide or an antibody binds Norovirus particle P domain at the H381 position of the P2 subdomain through the tyrosine residue .
  • the Norovirus P2 domain binding polypeptide specifically binds the P2 domain to the partial or complete exclusion of the binding of HBGA to the P2 domain.
  • the purified Norovirus P2 domain binding polypeptide competes for binding to the P2 domain with one or more HBGAs.
  • One use of the immunogenic compositions of the disclosure is to prophylactically treat a subject for Norovirus, such as in the early or late stages of infection, by inoculating an individual, particularly once a risk of developing disease from Norovirus infection has been indicated.
  • a "risk" means symptoms being presented or the individual having been present environment where Norovirus has been detected or is suspected of being present.
  • the anti-Norovirus compositions can be provided as a passive immunotherapy, intrabodies, and/or as humanized mAb agents for the detection and/or treatment of Norovirus related diseases.
  • the present disclosure provides for Norovirus therapeutics that can induce a specific immune response against Norovirus or provide passive immunity to Norovirus.
  • the term "antigen" is a molecule capable of being bound by an antibody or T-cell receptor.
  • An antigen is additionally capable of inducing a humoral immune response and/or cellular immune response leading to the production of B- and/or T- lymphocytes.
  • B -lymphocytes respond to foreign antigenic determinants via antibody production, whereas T-lymphocytes mediate cellular immunity.
  • the structural aspect of an antigen e.g., three dimensional conformation or modification (e.g., phosphorylation), which gives rise to a biological response is referred to herein as an "antigenic determinant" or "epitope.”
  • Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and usually at least 5 or 8- 10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include those methods described in Epitope Mapping Protocols (1996).
  • T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by H-thymidine incorporation by primed T cells in response to an epitope (Burke et ah , 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or by cytokine secretion.
  • immune response refers to a humoral (antibody mediated), cellular (mediated by antigen-specific T cells or their secretion products) or both humoral and cellular response directed against a protein, peptide, or polypeptide of the disclosure in a recipient patient.
  • Treatment or therapy can be an active immune response induced by administration of immunogen or a passive therapy effected by administration of antibody, antibody containing material, or primed T-cells.
  • Passive immunity refers to any immunity conferred upon a subject by administration of immune effectors including cellular mediators or protein mediators ⁇ e.g. , an polypeptide that binds to Norovirus protein).
  • An antibody composition may be used in passive immunization for the prevention or treatment of infection by organisms that carry the antigen recognized by the antibody.
  • An antibody composition may include antibodies or polypeptides comprising antibody CDR domains that bind to a variety of antigens that may in turn be associated with various organisms.
  • the antibody component can be a polyclonal antiserum.
  • the antibody or antibodies are affinity purified from an animal or second subject that has been challenged with an antigen(s).
  • an antibody mixture may be used, which is a mixture of monoclonal and/or polyclonal antibodies.
  • Passive immunity may be imparted to a patient or subject by administering to the patient immunoglobulins (Ig) or fragments thereof and/or other immune factors obtained from a donor or other non-patient source having a known immunoreactivity.
  • an antigenic composition can be administered to a subject who then acts as a source or donor for globulin, produced in response to challenge from the composition ("hyperimmune globulin"), that contains antibodies directed against Norovirus or other organism.
  • a subject thus treated would donate plasma from which hyperimmune globulin would then be obtained, via conventional plasma-fractionation methodology, and administered to another subject in order to impart resistance against or to treat Norovirus infection.
  • Hyperimmune globulins are particularly useful for immune-compromised individuals, for individuals undergoing invasive procedures or where time does not permit the individual to produce their own antibodies in response to vaccination. See U.S. Patents 6,936,258, 6,770,278, 6,756,361, 5,548,066, 5,512,282, 4,338,298, and 4,748,018, each of which is incorporated herein by reference in its entirety, for exemplary methods and compositions related to passive immunity.
  • epitopes and “antigenic determinant” are used interchangeably to refer to a site on an antigen to which B and/or T cells respond or recognize.
  • B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • T cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells.
  • T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or by cytokine secretion.
  • the presence of a cell-mediated immunological response can be determined by proliferation assays (CD4 (+) T cells) or CTL (cytotoxic T lymphocyte) assays.
  • proliferation assays CD4 (+) T cells
  • CTL cytotoxic T lymphocyte
  • the relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T-cells from an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.
  • the terms "antibody” or "immunoglobulin" are used interchangeably.
  • an antibody or preferably an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
  • a fusion protein with other proteins.
  • a method includes treatment for a disease or condition caused by a Norovirus pathogen.
  • embodiments include methods of treatment of Norovirus infection, such as hospital acquired nosocomial infections.
  • the treatment is administered in the presence of Norovirus antigens.
  • treatment comprises administration of other agents commonly used against bacterial infection, such as one or more antibiotics.
  • the therapeutic compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. Suitable regimens for initial administration and boosters are also variable, but are typified by an initial administration followed by subsequent administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a polypeptide therapeutic are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like. The dosage of the composition will depend on the route of administration and will vary according to the size and health of the subject.
  • compositions e.g., 2, 3, 4, 5, 6 or more administrations.
  • the administrations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9 , 10, 11, 12 twelve week intervals, including all ranges there between.
  • Certain aspects are directed to methods of preparing an antibody for use in prevention or treatment of Norovirus infection comprising the steps of immunizing a recipient with a vaccine and isolating antibody from the recipient, or producing a recombinant antibody.
  • An antibody prepared by these methods and used to treat or prevent a Norovirus infection is a further aspect.
  • a pharmaceutical composition comprising antibodies that specifically bind Norovirus and a pharmaceutically acceptable carrier is a further aspect that could be used in the manufacture of a medicament for the treatment or prevention of Norovirus disease.
  • a method for treatment or prevention of Norovirus infection comprising a step of administering to a patient an effective amount of the pharmaceutical preparation is a further aspect.
  • the antibody is a monoclonal antibody
  • the antibody is a polyclonal antibody.
  • Inocula for polyclonal antibody production are typically prepared by dispersing the antigenic composition (e.g., a peptide or antigen or epitope of Norovirus or a consensus thereof) in a physiologically tolerable diluent such as saline or other adjuvants suitable for human use to form an aqueous composition.
  • An immuno stimulatory amount of inoculum is administered to a mammal and the inoculated mammal is then maintained for a time sufficient for the antigenic composition to induce protective antibodies.
  • the antibodies can be isolated to the extent desired by well- known techniques such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual 1988).
  • Antibodies can include antiserum preparations from a variety of commonly used animals e.g., goats, primates, donkeys, swine, horses, guinea pigs, rats or man. The animals are bled and serum recovered.
  • An antibody can include whole antibodies, antibody fragments or subfragments.
  • Antibodies can be whole immunoglobulins of any class (e.g., IgG, IgM, IgA, IgD or IgE), chimeric antibodies, human antibodies, humanized antibodies, or hybrid antibodies with dual specificity to two or more antigens. They may also be fragments (e.g., F(ab')2, Fab', Fab, Fv and the like including hybrid fragments).
  • An antibody also includes natural, synthetic or genetically engineered proteins that act like an antibody by binding to specific antigens with a sufficient affinity.
  • a vaccine can be administered to a recipient who then acts as a source of antibodies, produced in response to challenge from the specific vaccine.
  • a subject thus treated would donate plasma from which antibody would be obtained via conventional plasma fractionation methodology.
  • the isolated antibody would be administered to the same or different subject in order to impart resistance against or treat Norovirus infection.
  • Antibodies are particularly useful for treatment or prevention of Norovirus disease in infants, immune compromised individuals or where treatment is required and there is no time for the individual to produce a response to vaccination.
  • An additional aspect is a pharmaceutical composition
  • a pharmaceutical composition comprising two of more antibodies or monoclonal antibodies (or fragments thereof; preferably human or humanized) reactive against at least two constituents of the immunogenic composition, which could be used to treat or prevent infection by Norovirus.
  • compositions and related methods particularly administration of an antibody that binds Norovirus or a peptide or consensus peptide thereof to a patient/subject, may also be used in combination with the administration of traditional therapies. These include, but are not limited to, the administration of one or more other antivirals.
  • a therapy is used in conjunction with antiviral treatment.
  • the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agents and/or a proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapeutic composition would still be able to exert an advantageously combined effect on the subject.
  • one may administer both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other.
  • compositions are administered to a subject. Different aspects may involve administering an effective amount of a composition to a subject.
  • an antibody that binds Norovirus or a peptide or consensus peptide thereof may be administered to the patient to protect against or treat infection by Norovirus.
  • an expression vector encoding one or more such antibodies or polypeptides or peptides may be given to a patient as a preventative treatment.
  • Such compositions can be administered in combination with an antibiotic.
  • Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.
  • the active compounds can be formulated for parenteral administration, e.g. , formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g. , formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the proteinaceous compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization or an equivalent procedure.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum- drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In certain embodiments, a vaccine composition may be inhaled (e.g. , U.S. Patent 6,651,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e. , the appropriate route and regimen.
  • Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • one or more antibodies or antibody-like molecules may be obtained or produced which have a specificity for a Norovirus. These antibodies may be used in various diagnostic or therapeutic applications described herein.
  • the term “antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE as well as polypeptides comprsing antibody CDR domains that retain antigen binding activity.
  • the term “antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and polypeptides with antibody CDRs, scaffolding domains that display the CDRs (e.g., anticalins) or a nanobody.
  • the nanobody can be antigen-specific VHH (e.g., a recombinant VHH) from a camelid IgG2 or IgG3, or a CDR- displaying frame from such camelid Ig.
  • VHH antigen-specific VHH
  • a recombinant VHH from a camelid IgG2 or IgG3, or a CDR- displaying frame from such camelid Ig.
  • the techniques for preparing and using various antibody-based constructs and fragments are well known in the art.
  • Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • Mini-antibodies or “minibodies” are also contemplated for use with embodiments.
  • Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region.
  • the oligomerization domain comprises self-associating oc-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds.
  • the oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
  • minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992); Cumber et al. (1992).
  • Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. (2003) describe "antibody like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods.
  • ABSiPs antibody like binding peptidomimetics
  • Alternative scaffolds for antigen binding peptides such as CDRs are also available and can be used to generate Norovirus-binding molecules in accordance with the embodiments.
  • CDRs antigen binding peptides
  • a person skilled in the art knows how to determine the type of protein scaffold on which to graft at least one of the CDRs arising from the original antibody. More particularly, it is known that to be selected such scaffolds must meet the greatest number of criteria as follows (Skerra, 2000): good phylogenetic conservation; known three- dimensional structure (as, for example, by crystallography, NMR spectroscopy or any other technique known to a person skilled in the art); small size; few or no post-transcriptional modifications; and/or easy to produce, express and purify.
  • the origin of such protein scaffolds can be, but is not limited to, the structures selected among: fibronectin and preferentially fibronectin type III domain 10, lipocalin, anticalin (Skerra, 2001), protein Z arising from domain B of protein A of Staphylococcus aureus, thioredoxin A or proteins with a repeated motif such as the "ankyrin repeat” (Kohl et al., 2003), the "armadillo repeat", the "leucine-rich repeat” and the "tetratricopeptide repeat”.
  • anticalins or lipocalin derivatives are a type of binding proteins that have affinities and specificities for various target molecules and can be used as Norovirus -binding molecules. Such proteins are described in US Patent Publication Nos. 20100285564, 20060058510, 20060088908, 20050106660, and PCT Publication No. WO2006/056464, incorporated herein by reference.
  • Scaffolds derived from toxins such as, for example, toxins from scorpions, insects, plants, mollusks, etc., and the protein inhibiters of neuronal NO synthase (PIN) may also be used in certain aspects.
  • toxins such as, for example, toxins from scorpions, insects, plants, mollusks, etc.
  • PIN protein inhibiters of neuronal NO synthase
  • Monoclonal antibodies are recognized to have certain advantages, e.g. , reproducibility and large-scale production. Embodiments include monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and chicken origin.
  • Humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
  • the term "humanized” immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin.
  • the non-human immunoglobulin providing the CDR's is called the "donor” and the human immunoglobulin providing the framework is called the "acceptor”.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • the strength with which an antibody molecule binds an epitope can be measured.
  • the affinity of an antibody may be determined by measuring an association constant (K a ) or dissociation constant(K d ).
  • Antibodies deemed useful in certain embodiments may have an association constant of about, at least about, or at most about 10 6 , 10 7 , 10 -8 , 10 9 or 10 10 M or any range derivable therein.
  • antibodies may have a dissoaciation constant of about, at least about or at most about 10 -6 , 10 -7 , 10 -8 , 10 -9 or 10 -10 . M or any range derivable therein.
  • a polyclonal antibody is prepared by immunizing an animal with a Norovirus polypeptide or a portion thereof in accordance with embodiments and collecting antisera from that immunized animal.
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat.
  • the choice of animal may be decided upon the ease of manipulation, costs or the desired amount of sera, as would be known to one of skill in the art.
  • antibodies can also be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom.
  • antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750, 172, and 5,741,957.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • Suitable adjuvants include any acceptable immuno stimulatory compound, such as cytokines, chemokines, cofactors, toxins, plasmodia, synthetic compositions or vectors encoding such adjuvants.
  • Adjuvants that may be used in accordance with embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL- 12, -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • MDP compounds such as thur-MDP and nor-MDP
  • CGP MTP-PE
  • MPL monophosphoryl lipid A
  • MPL monophosphoryl lipid A
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion is also contemplated.
  • MHC antigens may even be used.
  • Exemplary adjuvants may include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed My
  • BRM biologic response modifiers
  • CCM Cimetidine
  • CYP Cyclophosphamide
  • cytokines such as -interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7.
  • the amount of immunogen composition used in the production of antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen including but not limited to subcutaneous, intramuscular, intradermal, intraepidermal, intravenous and intraperitoneal.
  • the production of antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.
  • a second, booster dose (e.g. , provided in an injection), may also be given.
  • the process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g. , a purified or partially purified protein, polypeptide, peptide or domain, be it a wild-type or mutant composition. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • the methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. In some embodiments, Rodents such as mice and rats are used in generating monoclonal antibodies.
  • rabbit, sheep or frog cells are used in generating monoclonal antibodies.
  • the use of rats is well known and may provide certain advantages (Goding, 1986, pp. 60 61).
  • Mice e.g. , BALB/c mice
  • the animals are injected with antigen, generally as described above.
  • the antigen may be mixed with adjuvant, such as Freund's complete or incomplete adjuvant.
  • Booster administrations with the same antigen or DNA encoding the antigen may occur at approximately two- week intervals.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Generally, spleen cells are a rich source of antibody-producing cells that are in the dividing plasmablast stage. Typically, peripheral blood cells may be readily obtained, as peripheral blood is easily accessible.
  • B lymphocytes B lymphocytes
  • a panel of animals will have been immunized and the spleen of an animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 108 lymphocytes.
  • the antibody producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma producing fusion procedures preferably are non antibody producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • the culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse).
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • the individual cell lines could be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • Fragments of the monoclonal antibodies can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.
  • monoclonal antibody fragments can be synthesized using an automated peptide synthesizer.
  • monoclonal antibody fragments can be synthesized using an automated peptide synthesizer, or by expression of full-length gene or of gene fragments in E. coli.
  • fragments of a whole antibody can perform the function of binding antigens.
  • binding fragments are (i) the Fab fragment constituted with the VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment constituted with the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, 1989; McCafferty et al, 1990; Holt et al, 2003), which is constituted with a VH or a VL domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv) , wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, 1988; Huston
  • Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al., 1996).
  • Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. 1996). The citations in this paragraph are all incorporated by reference.
  • Antibodies also include bispecific antibodies.
  • Bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule (Holliger, P. & Winter, G. 1999 Cancer and metastasis rev. 18:411-419, 1999). Their use has been demonstrated both in the diagnostic field and in the therapy field from their capacity to recruit new effector functions or to target several molecules on the surface of tumor cells.
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger et al, PNAS USA 90:6444-6448, 1993), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
  • bispecific antibodies include those of the BiTETM technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides.
  • Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.
  • the citations in this paragraph are all incorporated by reference.
  • Bispecific antibodies can be constructed as entire IgG, as bispecific Fab'2, as Fab 'PEG, as diabodies or else as bispecific scFv. Further, two bispecific antibodies can be linked using routine methods known in the art to form tetravalent antibodies.
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against Norovirus, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by alternative engineering methods as described in Ridgeway et al, (Protein Eng., 9:616-621, 1996), which is hereby incorporated by reference.
  • compositions can be administered to a subject having, suspected of having, or at risk of developing a Norovirus related disease.
  • Therapeutic compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and boosters are also variable, but are typified by an initial administration followed by subsequent administrations.
  • compositions according to the present disclosure will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection.
  • a Norovirus- specific antibody that specifically binds an oligomer comprising a peptide having an amino acid sequence of SEQ ID NO: l can be administered into the cerebrospinal fluid of the brain or spine.
  • an immunogenic composition of the disclosure may be inhaled (e.g., U.S. Patent 6,651,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • compositions e.g., 2, 3, 4, 5, 6 or more administrations.
  • the administrations can be at 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11, 12 day or week intervals, including all ranges there between.
  • compositions are administered to a subject to treat Norovirus-related disease or condition.
  • an expression vector encoding one or more such antibodies or polypeptides or peptides may be given to a patient as a treatment.
  • Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • phrases "pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.
  • the active compounds of the present disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the form should be sterile and should be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the proteinaceous compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization (e.g., filter sterilization) or an equivalent procedure.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterilized solution thereof.
  • a pharmaceutical composition comprising antibodies that specifically bind an oligomer comprising a peptide having an amino acid sequence of the disclosure and a pharmaceutically acceptable carrier is a further aspect of the disclosure that can be used in the manufacture of a medicament for the treatment or prevention of a Norovirus-related disease or condition.
  • An additional aspect of the disclosure is a pharmaceutical composition
  • a pharmaceutical composition comprising one of more antibodies or monoclonal antibodies (or fragments thereof; preferably human or humanized) generated by using peptides having an amino acid sequence of the disclosure that specifically bind Norovirus. It is contemplated that in compositions of the disclosure, there is about 0.001, 0.01, 0.1, 1, 5, ⁇ g or mg to about 0.01, 0.1, 1, 5, 10 ⁇ g or mg of total polypeptide, peptide, and/or protein per ml.
  • the concentration of protein in a composition can be about, at least about or at most about 0.001, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg /ml, including all values and ranges there between.
  • the dose range is 0.01 to 500 mg/kg, 10 to 300 mg/kg, or 0.01 to 10 mg/kg.
  • About, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be a peptide having the amino acid sequence of the disclosure or antibody that specifically binds the same.
  • An effective amount of therapeutic or prophylactic composition is determined based on the intended goal, i.e., treatment or amelioration of a Norovirus -related disease.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the protection desired.
  • Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • Embodiments involve polypeptides, peptides, and proteins and immunogenic fragments thereof for use in various aspects described herein.
  • specific antibodies are assayed for or used in neutralizing or inhibiting Norovirus infection.
  • all or part of proteins described herein can also be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tarn et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.
  • Certain embodiments are related to peptides, antibodies, and antibody fragments for use in various embodiments of the present disclosure.
  • antibodies generated to a peptide comprising an amino acid sequence in a conformational epitope formed by surface-exposed loop clusters in the P domain in the capsid protein are utilized for specific binding to Norovirus.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • substitutions may be non-conservative such that a function or activity of the polypeptide is affected.
  • Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Proteins may be recombinant, or synthesized in vitro. Alternatively, a non- recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.
  • the term "functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region.
  • compositions there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
  • concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
  • polynucleotides encoding the proteins, polypeptides, or peptides described herein.
  • Polynucleotide sequences contemplated include those encoding antibodies to Norovirus, such as the P2 subdomain binding portions thereof.
  • polynucleotide refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences.
  • Polynucleotides may be single- stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.
  • the term "gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization).
  • this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • a nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein (see above).
  • nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof) that binds to Norovirus are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antibody or fragment thereof) that binds to Norovirus.
  • a polypeptide e.g., an antibody or fragment thereof
  • recombinant may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • nucleic acid segments regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.
  • Polypeptides may be encoded by a nucleic acid molecule.
  • the nucleic acid molecule can be in the form of a nucleic acid vector.
  • vector is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed.
  • a nucleic acid sequence can be "heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found.
  • Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • viruses bacteriophage, animal viruses, and plant viruses
  • artificial chromosomes e.g., YACs.
  • One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference).
  • Vectors may be used in a host cell to produce an antibody that binds Norovirus.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.
  • a "promoter” is a control sequence.
  • the promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • the particular promoter that is employed to control the expression of a peptide or protein encoding polynucleotide is not believed to be critical, so long as it is capable of expressing the polynucleotide in a targeted cell, preferably a bacterial cell. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • a vector in a host cell may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • ARS autonomously replicating sequence
  • the terms "cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • "host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • compositions discussed above Numerous expression systems exist that comprise at least a part or all of the compositions discussed above.
  • Prokaryote- and/or eukaryote-based systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC ® 2.0 from INVITROGEN ® and BACPACKTM BACULO VIRUS EXPRESSION SYSTEM FROM CLONTECH ® .
  • STRATAGENE ® 's COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system.
  • INVITROGEN ® which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
  • INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
  • a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
  • compositions described herein may be comprised in a kit.
  • a Norovirus antibody or immunogenic composition may be comprised in a kit in suitable container means.
  • the kit may be utilized for the treatment of Norovirus infection and/or for the prevention of Norovirus infection and/or for detection of Norovirus, including from a mammalian sample(s) and/or one or more environments.
  • the kit comprises certain monoclonal antibodies encompassed by the disclosure.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present disclosure also will typically include a means for containing the Norovirus antibody or immunogenic composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the Norovirus antibody or immunogenic compositions may also be formulated into a syringeable composition.
  • the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
  • kits of the present disclosure will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • the kit comprises one or more apparatuses and/or reagents for obtaining a sample from an individual and/or processing thereof.
  • the contact area with several hydrogen bonds and hydrophobic interactions is quite extensive with a buried surface area of -700 A ° 2" (FIG. 7).
  • a prominent stabilizing interaction is the stacking interaction between the tyrosine residue CDRL1 and the histidine residue (H381) of the P2 subdomain.
  • the inventors have successfully isolated a panel of human monoclonal antibodies from an individual infected with NV, and characterized their ability to block HBGA binding. Several of these antibodies clearly interact with NV with high affinity and block HBGA binding effectively.
  • Human noroviruses cause acute gastroenteritis worldwide. They evolve with the periodic emergence of new epidemic strains based on antigenic variations and differential glycan binding specificities that lead to sequence and structural changes in the P domain of the NoV capsid protein. Histo blood group antigens (HBGAs) serve as susceptibility and cell attachment factors to HuNoVs. Recent studies show that the presence of antibodies that block virus-HBGA interactions is associated with protection against illness and thus act as putative neutralization antibodies (NAbs). Although the structural basis of HBGA binding is well characterized, there are no structural studies to explain the basis of antigenic variation and how these NAbs block HBGA binding in HuNoVs.
  • HBGAs Histo blood group antigens
  • B cells were isolated from a person challenged with the prototype Norwalk virus (NV) and screened for antibodies that block HBGA interactions with NV VLPs.
  • Fab fragments from one of the HBGA-blocking monoclonal antibodies (512) were generated for crystallographic studies of NV P domain-Fab 512 complex. The structural studies reveal that Fab binds to the loops located on the top of the P domain without inducing any significant conformational changes in the P domain.
  • the Fab binding site is in close proximity to the HBGA binding site thereby blocking access to the HBGAs.
  • the identified Fab binding loops are known to undergo structural variations among other NoVs genotypes, which could explain the molecular mechanism underlying antigenic variation.
  • the complementarity determining regions (CDRs) of the Fab that interact with the P -domain are also clearly delineated which could be used to for the structure-based design and optimization of scaffolds that can block HBGA binding across the genotypes.
  • Such scaffolds in turn can be used in the development of vaccines and/or antivirals against noroviruses.
  • Human noroviruses are the leading cause of viral gastroenteritis. They are associated with almost a fifth of all cases of acute gastroenteritis worldwide (Ahmen, et al., 2014). It is estimated that approximately 200,000 children under the age of 5 years die annually from HuNoV infections (Patel, et al., 2008). Currently there are no licensed vaccines or antivirals to treat the disease, although vaccine candidates are in the pipeline (Treanor, et al., 2014; Atmar, et al., 2011). Development of efficient vaccines is limited by lack of understanding of the immune correlates of protection and rapid evolution of NoVs based on antigenic variations and differential glycan binding.
  • Noroviruses are non-enveloped positive strand RNA viruses belonging to the family Caliciviridae. They are phylogenetically classified into at least six genogroups (GI-GVI) with each genogroup divided into several genotypes. Genogroups GI, Gil and GIV contain human pathogens (Green, et al., 2000; Ramani, et al., 2014). The prototype Norwalk virus (NV) is classified as genogroup I genotype 1 (GI. l). NoVs belonging to genotype GII.4 are the most prevalent and are associated with -70% of all HuNoV infections (Lindesmith, et al., 2011).
  • VLPs virus like particles
  • VPl is composed of two principal domains, the shell (S) domain, which is involved in the formation of icosahedral shell, and the protruding (P) domain that projects out from the shell (Prasad, et al, 1999).
  • the P domain is further divided into PI and P2 subdomains, with the latter being an insertion in the PI subdomain.
  • the P2 subdomain is the least conserved and is implicated in strain diversity, differential HBGA binding and antigenicity (Singh, et al, 2015' Shanker, et al, 2014).
  • HuNoVs are suggested to evolve through a coordinated interplay between differential HBGA binding specificities and antigenic variations that allow emerging strains to escape host immunity. Differential HBGA binding has been previously well characterized in both GI and Gil HuNoVs (Huang, et al, 2005; Shanker, et al, 2011).
  • IgA 512 was shown to be genotype specific; it binds to the P domain of GI. l NV, and effectively blocks HBGA binding to NV VLP.
  • Fab 512 and NV P domain complex were assessed for crystallographic studies.
  • biotinylated P domain was immobilized on a streptavidin biosensor and titrated against serial dilutions of Fab 512.
  • the P domain-Fab 512 complex crystals diffracted to -2.3A and the structure was determined in the space group P6 5 22, with one P domain-Fab complex in the crystallographic asymmetric unit.
  • the structure of the complex was determined using molecular replacement techniques and refined with a final R fac and R free values of 18% and 21% respectively (Table 1).
  • the P domains related by crystallographic 2-fold symmetry associate to form a dimer, as typically found in the NV capsid and other NoV P domain structures, with each of the dimeric subunits interacting separately with a Fab 512 molecule (Fig. 13).
  • the Fab recognizes and interacts with a conformational epitope in the P2 subdomain.
  • Superposition of the unbound and Fab 512-bound P domain structures showed that Fab binding does not alter the overall structure of the P domain (r.m.s.d. of 0.5 A) but Fab binding does induce local conformation changes in some of the loop regions.
  • Tafele 1 Data prscess.1 ⁇ 2g ais.fi ReSiieiiieai statistics
  • the overall structure of the bound Fab 512 is similar to other structurally characterized Fabs.
  • the constant (CH and CL) and variable (VH and VL) domains of the heavy and light chains exhibit a typical immunoglobulin fold.
  • the CH and CL interact closely with one another as do the VL and VH.
  • the three hypervariable complementarity determining regions (CDRs) from each heavy (CDR- HI, H2 and H3) and light chains (CDR- LI, L2, L3) in the variable domains are oriented facing the P2 subdomain.
  • the CDR loops of Fab 512 are of varying lengths, with CDRH3 and CDRL1 being the longest, each consisting of 17 residues. Although the length of CDRH3 with 17 residues is typical, the 17-residue length of CDRL1 is unusual, and analysis of the interfacial interactions between P domain and Fab shows that CDRL1 plays a dominant role in antigen recognition.
  • the paratope of Fab 512 comprises three of the six CDRs including CDRL1 (residues 24-40) and CDRL3 (residues 96 -103) in the light chain and CDRH3 (residues 97-113) in the heavy chain (FIG. 14A).
  • CDRL1 deoxyribonucleic acid
  • CDRL3 deoxyribonucleic acid 3
  • CDRH3 CDRH3
  • CDRL1 makes the most extensive interaction with the P domain. Its residues Q27, S28, L30, K32 and K35 contribute to eight hydrogen bonding interactions with the P domain residues N346, T348, D350 and F352 in loop Q and residues N394, G396 and S398 in loop U. CDRL1 also contributes to several water-mediated hydrogen bonding interactions. CDRL3 participates in the epitope recognition through its residues Y98 and 1100. Y98 makes two hydrogen bonds, one with residue T348 in loop U and another with H381 in loop T. 1100 is involved in water-mediated hydrogen bonding interaction with residue S380 in loop T.
  • CDRH3 is the lone CDR from the heavy chain of Fab 512 that interacts with the P domain. Interactions involve its residues, Y107 and D108. Y107 is involved in hydrophobic and water mediated hydrogen bond interaction with residue S383 in loop T of P domain and D108 makes two direct hydrogen bonds with residue S383 on the T loop of P domain (FIG. 14B).
  • the P domain-Fab complex structure also exhibits interactions that contribute significantly to surface complementarity.
  • the side chain of K32 from CDRL1 buries itself into a narrow, -8A deep pocket on the surface of P domain, contributing to a network of hydrogen bonding interactions. While the main chain amide group of K32 hydrogen bonds with the main chain carbonyl group of T348 on the rim of the pocket, its side chain hydrogen bonds with the main chain carbonyl groups of F352, N394 and G396 lying at the bottom of the pocket (FIG. 15A).
  • This loop shifts by as much as 10A (at maximum Ca divergence) to make favorable interactions with CDRL1 including hydrogen bonding interactions described above involving K32 of CDRL1 (FIG. 15B).
  • the orientation of the H381 sidechain is flipped as compared to its orientation in the unbound P domain structure (FIG. 15D). This flipping ensures the H381 side chain does not sterically hinder binding of Fab 512 and allows it to participate in favorable intramolecular stacking interactions with the side chain of P382 of the P domain.
  • HuNoVs are unique among viral pathogens in exploiting the genetically-controlled polymorphic nature of the HBGAs among host populations for their sustained evolution (Huang, et al., 2005; Lindesmith, et al., 2008)).
  • the distally located P2 subdomain can evolve to escape neutralization and differentially interact with HBGAs as underscored by recent studies that show HBGA-blocking antibodies confer protection against HuNoV infection (Reeck, et al., 2010; Bok, et al., 2011).
  • HuNoV interactions with HBGAs the understanding of the mechanism by which a human antibody blocks HBGA binding is limited.
  • the crystallographic structure of the NV GI.
  • IgA 512 P domain in complex with the Fab of a human IgA 512 monoclonal antibody addresses key questions such as how a blockade antibody recognizes HuNoV, what is the mechanism of HBGA blockade, and how sequence alterations allow other genotypes to escape neutralization.
  • the IgA 512 was selected from a panel of HBGA blocking antibodies obtained by generating hybridomas from B cells isolated from an individual challenged with GI.l NV. Both IgG and IgA antibodies were identified.
  • An IgA antibody was chosen for the structural studies because of the important role of IgA, compared to IgG, in conferring mucosal immunity. The subnanomolar binding affinity of this antibody together with its HBGA blockade activity in vitro, is suggestive of its high potency in virus neutralization.
  • IgA 512 recognizes a conformational epitope formed by the P2 subdomain loops -
  • the crystal structure of the Fab 512 in complex with the NV P domain shows that Fab recognizes a conformational epitope comprised of residues from the solvent-exposed loops in the distal portion of the P2 subdomain.
  • Involvement of the surface loops in antibody recognition is acommon feature as observed in many antigen-antibody structures.
  • the distal surface of the P2 subdomain consists of 6 loops that project out into the solvent, which can be grouped into three clusters 1-3 (FIG. 18A). Despite sequence changes, differences in their lengths and orientations, these loops are similarly clustered in GII.4 (FIG.
  • Residues from clusters 1 and 2 in GI.l constitute the antigenic site recognized by IgA 512 (FIG. 18A).
  • IgA 512 specifically recognizes clusters 1 and 2
  • other blockade antibodies can recognize residues in other clusters as suggested by previous biochemical studies characterizing such antibodies in GI and GII.4.
  • residues in the A and B loops in cluster 3 have been identified as important for binding blockade antibodies (Chen Z et al. J Virol 2013;87:9547-57).
  • CDRL1 plays a dominant role in antigen recognition -
  • a rather unusual feature of IgA 512 is the dominant involvement of CDRL1 in antigen recognition, providing a unique perspective into antibody diversity and antigen interactions.
  • CDRH3 encoded by the highly diverse D-JH joining genes plays a dominant role because of the inherent sequence diversity and consequent conformational variability.
  • the H3 loop is also a common site for somatic hypermutations, allowing affinity maturation of the antibodies (Tsuchiya, et ah, 2016; Shirai, et al., 1999).
  • IgA 512 five out of eight residues in the CDRs that interact with P domain are from CDRL1.
  • the length of the CDRL1 varies between 10 and 17 residues, with the majority of the antibodies exhibiting a loop length of 11 residues.
  • CDRL1 is 17 residues long. Despite its unusual length, it exhibits the expected canonical conformation.
  • the CDRH3 is also 17 residues long and is within the expected range of 10-30 residues. The general expectation is that H3 loops with longer lengths (>14) play a predominant role in antigen specificity, whereas in those with shorter lengths, antigen interactions involve other loops.
  • H3 loop is positioned slightly away from the P2 subdomain, with just two of its residues interacting with the P2 subdomain.
  • CDRL1 The other non-H3 CDRs are of normal lengths with canonical conformations as observed in other antibody structures. With the exception of two residues in the CDRL3, residues from other CDRs do not participate in antigen recognition. CDRL1, together with L3 and H3 residues, provide the complimentary residues for optimal hydrogen bond and hydrophobic interactions with the loop residues of the P2 subdomain, as well as appropriate topographical features to enhance the surface complementarity with the P2 subdomain consistent with the observed binding affinity in the low nanomolar range. It remains to be seen whether the dominant role of CDRLl observed with IgA 512 is a common feature in HuNoV blockade antibodies.
  • HBGA blockade by Fab 512 is by steric hindrance - HBGA blockade by an antibody potentially can occur in a number of ways, including directly competing for the HBGA binding site, allosterically disrupting the HBGA binding site by inducing conformational changes in the P domain, or by sterically masking the HBGA binding site.
  • the crystallographic studies show that in the case of IgA 512, the mechanism of HBGA blockade is principally through steric hindrance.
  • the Fab 512 binds to the NV P domain without affecting either the dimeric conformation of the P domain or the structural integrity of the HBGA binding site.
  • the HBGA binding site is located in a shallow depression on the distal surface of the P2 subdomain surrounded by clusters of loop regions.
  • the HBGA binding sites in both GI and Gil are surrounded by loop regions.
  • the majority of the residues mapped by biochemical studies as being critical for blockade antibody binding are outside of the primary HBGA binding site, whether it is the ⁇ Gal binding site in the case of GI or the a Fuc site, as seen in GII.4 HuNoVs.
  • most of the HBGA blocking mAbs characterized thus far are genotype specific and do not cross react, even within the same genogroup, similar to IgA 512, suggesting that these mAbs also primarily interact with the loop regions that are prone to genotypic alterations.
  • HBGA blocking polyclonal antibodies from the HuNoV-infected individuals have shown cross -reactivity (Czako, et ah, 2015), many derived mAbs, such as IgA 512, are genotype-specific (Lindesmith, et al., 2013; Payne, et al., 2015).
  • IgA 512 is highly specific for GI.
  • the H381 residue in the T loop of NV P domain is critical for IgA 512 binding as it is involved in multiple stabilizing interactions with the antibody and is not conserved in other GI genotypes. While this residue in GI. l is far removed from the HBGA binding site, because of the conformational changes, the structurally corresponding residue S391 in GI.7 becomes a part of the primary HBGA binding site (Shanker, et al, 2014) clearly illustrating a coordinated interplay between antigenic variation and HBGA binding in the evolution of No Vs.
  • HBGA blockade mAbs tend to be genotype-specific
  • one possibility is to use a cocktail of such antibodies, or to design antibody scaffolds with a smaller footprint such as single-chain antibodies or even small molecule mimics that specifically target highly conserved HBGA binding site. Further studies are clearly required to explore such a possibility.
  • variable domain sequences of IgA 512 and synthesis of expression-optimized genes was done as described previously (Sapparapu et al. submitted).
  • the VH domain was cloned as an EcoRI / Hindlll fragment into pHC-huCglFab expression vector.
  • the VL domain was cloned as a Bglll I Notl fragment into pML-huCk kappa expression vector (McLean, et al., 2000).
  • Recombinant antibodies were expressed transiently in Expi293F cells by cotransfection of equal amount of heavy and light chain plasmid DNA using ExpiFectamine 293 transfection reagent (Life Technologies).
  • P domain-Fab 512 complex formation and crystallization Purified P domain (mw 32kd) and Fab 512 (mw 50kd) proteins were mixed in a 1: 1 molar ratio in the P domain storage buffer and incubated for 2-4 hours at 4°C. The mixture was run through the S75pg 16/60 gel filtration column and the peak corresponding to the complex (assessed by peak shift compared to P domain by itself) was collected. The complex eluted at an mw of approximately 160kd corresponding to a P domain dimer bound to two Fab molecules. SDS- PAGE confirmed the presence of both the proteins in the complex peak. The peak fractions were then pooled and concentrated to lOmg/ml for crystallization trials.
  • Crystallization screening using hanging-drop vapor diffusion method at 20°C was set up by nanoliter handling system Mosquito (TTP Lab Tech) with commercially available crystal screens.
  • Initial crystals were small and diffracted to > 3.5 A.
  • the initial crystallization conditions were further optimized based on ionic strength, pH and precipitant concentrations, and microseeding technique was employed to obtain larger well diffracting crystals. Crystals measuring 0.1-0.2 mm were obtained in 1- 2 weeks. The crystals were soaked in the reservoir solution containing 20% glycerol as cryoprotectant followed by flash freezing in liquid nitrogen.
  • Diffraction, data collection and structure determination Diffraction data for the P domain-Fab 512 crystals were collected on the 5.0.1 beamline at Advance Light Source Berkeley. Diffraction data were processed using IMOSFLM (Battye, et al., 2011). Space group was confirmed using POINTLESS program incorporated in the PHENIX suite (Adams, et al., 2002). Initial electron density map was obtained by molecular replacement (MR) using the previously published GI. l P domain structure (PDB ID: 2ZL5) as the starting model using program PHASER (McCoy, et al., 2007) in the CCP4i suite (Collaborative Computational Project, et al., 1994).
  • MR molecular replacement
  • Norwalk virus the prototype of human noroviruses (NoVs) was the first virus identified in 1972 as a causative agent for acute gastroenteritis (Kapikian, 2000). NoVs are the leading cause of epidemic acute and sporadic cases of gastroenteritis responsible for about 19-21 million cases of infection leading to >70,000 hospitalizations and about 800 deaths annually in the U.S. (Hall, et al., 2013). NoVs recently surpassed rotaviruses as the leading cause of pediatric non-bacterial gastroenteritis after the introduction of vaccines against rotaviruses (Payne, et al., 2013). The infection is typically self-limiting, lasts for 1-3 days, and is characterized by diarrhea, vomiting, nausea, stomach pain and fever, with more severe complications and chronic disease in the immunocompromised. Therapy involves rest and rehydration, and no specific therapeutic agent is currently available.
  • NoVs members of the Caliciviridae family, are non-enveloped and contain a positive-sense, non-segmented single stranded RNA genome enclosed by a protein capsid.
  • the genome codes for three open reading frames (ORF), with the first ORF coding for six non-structural proteins involved in viral transcription and replication.
  • the second and third ORFs encode virus protein (VP1) and VP2, respectively.
  • VP1 is a major capsid -60 kDa protein and can self-assemble into virus-like particles (VLP) that resemble native virions both morphologically and antigenically (Jiang, et al., 1992).
  • the viruses are classified into at least six genogroups (GI, Gil, GUI, GIV, GV and GVI), based upon the sequence of VP1 (Ramani, et al., 2014).
  • the genogroups are further subdivided into genotypes, with GI and Gil accounting for the most diversity with 9 and 22 genotypes, respectively.
  • GI and Gil NoVs are responsible for the majority of human infections, with the genotype GII.4 responsible for most.
  • Human susceptibility to 58 NoVs depends on the expression of histo-blood group antigens (HBGAs) on the intestinal epithelial cells (Marionneau, et al., 2002; Lindesmith, et al., 2003; Hutson, et al., 2003).
  • PBMC peripheral blood mononuclear cell
  • Cells from the positive wells were cloned biologically by sorting single cells into 384-well plates using a FACSAria III fluorescence activated cell sorter (Becton Dickinson), cultured for about 14 days 117 and screened for antibody production.
  • a FACSAria III fluorescence activated cell sorter Becton Dickinson
  • PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) and sequenced directly using an ABI3700 automated DNA sequencer without cloning. Heavy chain or light chain antibody variable region sequences were analyzed using the EVIGT/V- Quest program (Brochet, et al., 2008; Giudicelli, et al., 2011). The analysis involved the identification of germline genes that were used for antibody production, location of complementary determining regions (CDRs) and framework regions (FRs) as well as the number and location of somatic mutations that occurred during affinity maturation.
  • CDRs complementary determining regions
  • FRs framework regions
  • variable domains For expression of recombinant forms of the antibody clones, the nucleotide sequences of variable domains were optimized for mammalian expression and synthesized (Genscript).
  • the heavy chain fragments were cloned as EcoRI/Hindlll fragments into 138 pML-huCGl or pML- huCAl vectors for expression of ⁇ or al chains, respectively (Mclean, et al., 2000).
  • the light chains were cloned as Bglll/NotI fragments into pML-huCk or pML-huCL vectors for ⁇ or ⁇ chains, respectively.
  • Transfection was done using ExpiFectamine 293 transfection reagent (Life Technologies) according to the manufacturer's protocols. After 7 days of culture, the supernatants were clarified by centrifugation and filtered using 0.4- ⁇ pore size filter devices. Antibodies were harvested from the supernatants by affinity chromatography on HiTrap KappaSelect or LambdaSelect columns (Life Technologies) as previously described (Aiyegbo, et al., 2013). Antibodies eluted from affinity columns were concentrated using Amicon centrifugal filters (Millipore). Purified antibodies were resolved on polyacrylamide gels under reducing or non-reducing denaturing conditions and stained with Coomassie Blue reagent.
  • Antibodies used as control reagents Polyclonal rabbit serum raised against NoV VLPs was obtained as a positive control for detection of VLPs coated on ELISA plates. This immune sera were generated by hyperimmunization of rabbits with NV VLPs as previously described (Jiang, et al., 1992). The inventors also prepared purified immunoglobulin from murine hybridoma cells secreting the mAbs 8812 or 3901. MAbs 8812 and 3901 were included in some receptor experiments as positive and negative controls for inhibition of NV VLP binding to receptor, based on previously determined activities (Hutson, et al., 2003).
  • VLPs - VLPs representing different norovirus genogroups (GI and Gil) and genotypes (GI. l, NC_001959; GI.2, FJ515294; GI.4, GQ413970; GI.6, KC998959; GI.7, JN005886; GI.8, GU299761; GII.4; EU310927) were generated and purified as previously described (Kou, et al., 2015).
  • capsid proteins (VP1 and VP2) were expressed in SF9 insect cells (2.75xlO A 6 cells/mL of Grace's insect cell media) from recombinant baculovirus expression vectors, and NoV VLPs were purified from culture supernatants on a cesium chloride gradient (Jiang, et al., 1992). Structural integrity and purity of the VLP preparations were confirmed by electron microscopy of negatively stained VLPs (1.0% ammonium molybdenate (Sigma-Aldrich; St. Louis, MO), 180 pH 6.0) on carbon coated grids and by Western blot, respectively.
  • the inventors also generated a GI.1 VLP (designated CT303) in which the P domain was deleted by mutagenesis of the VP1 gene construct (Bertolotti-Ciarlet, et al., 2002).
  • a second mutated GI. l VLP was prepared with the point mutation W375A that was previously determined to ablate HBGA binding (Choi, et ah, 2008).
  • VLP binding assay - Binding characterization of purified antibodies to NoV VLPs was carried out by ELISA.
  • NoV VLPs were suspended in PBS at 1 ⁇ g/mL and coated in microwell plates (Nunc) for 16 h at 4 °C, and the wells were blocked with 5% skim milk and 2% goat serum in PBS-Tween.
  • Purified antibodies were diluted serially in PBS and added to the ELISA plates. The bound antibodies were detected using alkaline phosphatase conjugated goat anti-human ⁇ or ⁇ chain antibodies (Southern Biotech).
  • the genotype specificity of antibody binding was determined by direct ELISA, as described above, with the following modifications: VLPs were coated at 10 ⁇ g/mL and antibodies were used at a concentration of 20 ⁇ g/mL. Plates were developed using ultra- TMB reagent (Pierce ThermoFisher; Rockford, IL), following the manufacturer's protocol, and optical density as read at 450 nm using a SpectraMax M5 plate reader.
  • P domain dimer specific binding assay The inventors prepared purified recombinant P domain dimeric protein, as previously described (Choi, et al., 2008). Briefly, a NV P domain construct was expressed in E. coli (Novagen) and purified by affinity chromatography, followed by size exclusion chromatography. We tested binding of each of the mAbs to P domain dimer by direct antigen ELISA, using the same protocol as described above for the VLP binding assay.
  • Human mAbs were diluted to 1 ⁇ g/mL in blocking solution (1% wt:vol, Kroger non-fat dried milk in IX phosphate buffered saline). Two NV- reactive murine monoclonal antibodies (mAb 3901 and mAb 8812) and a Norwalk-reactive rabbit polyclonal were used as positive controls for detection of VPl. Blots were incubated overnight at 4°C. Bound antibodies were detected using either an anti-human Ig (A, G, M)- HRP, anti-mouse-HRP, or anti-rabbit-HRP conjugate antibody (Southern Biotech; Birmingham, AL). Blots were developed by chemiluminescence using West Pico HRP substrate (Pierce ThermoFisher; Rockford, IL) following the manufacturer's instructions.
  • HBGA blocking assay Disruption of interaction between VLP and HBGAs was used as a surrogate assay for measuring NoV neutralization by human monoclonal antibodies. Pre-existing titer of HBGA blocking antibodies is correlated with protection from NoV gastroenteritis (Reeck, et al., 2010; Atmar, et al., 2011). An HBGA blocking assay was carried out as previously described (Reeck, et al., 2010). Briefly, biotin-polyacryamide (PAA)-blood group antigen conjugates (Glycotech, Rockville, MD) were immobilized on neutravidin-coated plates (Thermo Scientific).
  • PAA biotin-polyacryamide
  • VLPs were mixed with serial dilutions of antibodies, and the complexes were added to the glycan-coated microtiter plates.
  • the relative amount of VLP bound to HBGAs was determined using rabbit anti-NoV antiserum followed by horseradish peroxidase-conjugated goat anti-rabbit (Southern Biotech).
  • H type 1 H type 1
  • H type 2 H2-PAA-biotin
  • H type 3 H3-PAA-biotin
  • a trisaccharide tri-A-PAA-biotin
  • Lewis(y) Lewis(y)-PAA-biotin
  • Plates were developed using ultra- TMB reagent (Pierce ThermoFisher; Rockford, IL), following the manufacturer's protocol, and optical density as read at 450 nm using a SpectraMax M5 plate reader.
  • Hemagglutination inhibition assay Hemagglutination inhibition assays were performed as described previously (Czako, et al., 2012). In brief, Human type O erythrocytes were collected from a healthy adult in Alsever's buffer, washed twice in Dulbecco's phosphate -buffered saline (PBS) without Ca2+ or Mg2+, and pelleted by centrifugation at 500xg for 10 min at 4 °C.
  • PBS Dulbecco's phosphate -buffered saline
  • Monoclonal antibodies (mAb; starting concentration 60 ⁇ g/mL for human mAb and 8.5 ⁇ g/mL for murine 245 8812) were diluted initially 1: 10 in PBS with 0.85% saline (pH 5.5), and then serially 2-fold diluted.
  • Four hemagglutination units ( ⁇ 2 ng) of Norwalk virus VLPs were mixed with the diluted monoclonal antibodies and incubated at room temperature for 30 min.
  • the VLP-mAb mixture was added to an equal volume of 0.5% washed type O erythrocytes in 0.85% saline (pH 6.2) and incubated for 2 h at 250 °C.
  • the HAI titer was determined by identifying the reciprocal of the highest dilution of mAb that inhibited hemagglutination by the VLPs.
  • Competition-binding ELISA analysis - Competition-binding ELISAs were carried out to determine whether the hmAbs we generated bound distinct or shared epitopes in the NV capsid protein. Briefly, each mAb was used to coat a 96-well microtiter plate (Greiner Bio-One; Monroe, NC) at a concentration of 2 ⁇ g/mL in carbonate coating buffer at 4°C overnight.
  • Norwalk VLPs (100 ng/mL) were incubated with serial dilutions of each hMAb, ranging from 6.25 ⁇ g/mL to 250 ⁇ g/mL in assay buffer [1% non-fat dried milk (NFDM) in IX PBS, w/v], for 2 hours at 37 °C.
  • assay buffer [1% non-fat dried milk (NFDM) in IX PBS, w/v]
  • NFDM non-fat dried milk
  • IX PBS IX PBS, w/v
  • VLP/mAb preparations were added to the mAb-coated microtiter plate and plates were incubated for 2 hours at 37°C.
  • Bound VLPs were detected using a rabbit anti-NV polyclonal antibody (1/10,000 in assay buffer; 2 hours at 37°C) followed by a commercial goat anti-rabbit-HRP conjugate antibody (Southern Biotech; 1/7500 in assay buffer; 45 minutes at 37°C). Plates were developed using ultra-TMB reagent (Pierce ThermoFisher; Rockford, IL), following the manufacturer's protocol, and optical density as 267 read at 450 nm using a SpectraMax M5 plate reader. Readings from duplicate wells were averaged.
  • the percent competition for each competitor hMAb was calculated relative to the antigen-only control. MAbs were judged to compete for binding to the same site if maximum binding of the competing mAb was reduced to ⁇ 25% of its un-competed binding. A level of 25-50% of its un competed binding was considered intermediate competition.
  • Polyclonal secondary antibodies instead of monoclonal antibodies, were used to minimize any differences in sensitivity of the secondary antibody to gamma or alpha chains and confirmed that the affinities of secondary antibodies did not differ measurably.
  • the inventors were able to obtain a panel of seven IgG (1A8, 2L8, 3123, 4E7, 4123 from Donor 1 and NV1, NV48 from Donor 2) and seven IgA (2J3, 313, 4B 19, 4C10, 512 from Donor 1 and NV41, NV56 from Donor 2) clones.
  • the proper molecular assembly of IgG and dimeric IgA was confirmed by resolving antibodies on SDS- PAGE gels and staining with Coomassie Blue reagent (FIG. 23).
  • IgA antibodies are more potent than IgG for receptor blocking - Interpretation of the curves for Ig binding to VLPs was conducted after normalizing for the differing molarity of binding sites of IgG and dimeric IgA. IgA antibodies as a class appeared to have a lower affinity for binding in the VLP binding assays when compared with IgG. Interestingly, however, this class distinction was not apparent in the assays to detect antibody mediated blocking of VLP binding to glycan. These data suggest that even lower affinity IgA antibodies can mediate potent blocking activity (FIG. 20).
  • the VP1 protein has two major domains, the highly conserved shell domain and the highly variable protruding (P) domain.
  • P protruding
  • the inventors also tested NV VLPs with ablated HBGA binding through introduction of a point mutation (W375A) in the HBGA binding domain.
  • MAbs from a NV-infected individual bind nonlinear epitopes -
  • Murine mAbs 3901 or 8812 have been described previously to bind to linear or nonlinear epitopes, respectively, and were used as controls in this experiment (Hardy, et al., 1996).
  • MAbs recognize at least 3 overlapping epitopes in VP1 - Epitope binning was carried out by competition-binding ELISA. MAbs were assessed in a pairwise manner for their ability to inhibit binding of each other to NoV VLPs by ELISA (FIG. 22C). The observed patterns of competition-binding suggest that most of the mAbs bind to one major antigenic site. However, a few mAbs (2L8 IgG and 3123 IgG) failed to inhibit capture of NV VLPs by other mAbs (4B 19 IgA, 4C10 IgA, 4123 IgG, 512 IgA).
  • IgG or IgA versions of representative blocking antibodies using mammalian cell recombinant expression of isotype- switch variant Ig molecules.
  • the inventors synthesized cDNAs coding for the variable domains after optimizing the sequence of the genes computationally for expression in mammalian cells.
  • the heavy chain antibody variable genes were cloned in expression vectors for expression as ⁇ or a chain.
  • the light chain antibody variable genes were cloned in expression vectors for expression as ⁇ or ⁇ chains.
  • Recombinant polymeric IgA was obtained by co-expression of joining (J) chain along with the heavy and light chains.
  • Electrophoresis of purified proteins on SDS-PAGE gels under non-reducing conditions confirmed the correct assembly of IgG and dimeric IgA (FIG. 25).
  • each set of antibodies was tested in the binding and blocking assays.
  • the inventors calculated half-maximal effective concentrations (EC 50) at which binding or blocking occurred.
  • EC 50 half-maximal effective concentrations
  • the concentration at which half-maximal binding (EC50) or inhibition (IC50) occurred was calculated from non-linear regression analysis.
  • the ratio of IC50 to EC50 suggests that more IgG is needed for blocking activity compared to mlgA or dlgA for all the three antibodies compared.
  • Table 5 Hemagglutination inhibition activity for recombinant isotype switch variants. Hemagglutination inhibition assays were performed as described previously (Czako, et al., 2012). In brief, human type O erythrocytes were collected from a healthy adult in Alsever' s buffer, washed twice in Dulbecco' s phosphate-buffered saline (PBS) without Ca2+ or Mg2+, and pelleted by centrifugation at 500 x g for 10 min at 4 °C.
  • PBS Dulbecco' s phosphate-buffered saline
  • Monoclonal antibodies (mAb; starting concentration 60 ⁇ g/mL) were diluted initially 1: 10 in PBS with 0.85% saline (pH 5.5), and then serially 2-fold diluted.
  • Four hemagglutination units ( ⁇ 2 ng) 683 of Norwalk virus VLPs were mixed with the diluted monoclonal antibodies and incubated at room temperature for 30 min.
  • the VLP-mAb mixture was added to an equal volume of 0.5% washed type O erythrocytes in 0.85% saline (pH 6.2) and incubated for 2 h at 4 °C.
  • the HAI titer was determined by identifying the reciprocal of the highest dilution of mAb that inhibited hemagglutination by the VLPs.
  • Blocking activity of purified, serum derived IgA antibodies was recently described, and our group recently identified serum IgA and salivary IgA antibodies as novel correlates of protection from NoV gastroenteritis (Atmar, et ah, 2015; Ramani, et al., 2015; Lindesmith, et al., 2015).
  • the inventors isolated a panel of hmAbs with potent NoV-HBGA blocking activity, representing IgA and IgG isotypes, from an immune individual following experimental virus challenge. The features of these antibodies reveal new aspects of antibody-mediated NoV inhibition.
  • dimeric IgAs exhibited enhanced potency for blocking compared to matched monomeric IgA or IgG counterparts. Most likely, this finding is due to the large molecular weight of dimeric IgA, which probably facilitates a more profound receptor blocking capacity.
  • the NoV receptor-blocking antibodies did not possess any extreme genetic features. Diverse antibody variable genes were used, and the level of somatic mutation observed was typical of that found in human memory B cells (Briney, et al., 2012a; Briney, et al., 2012b).
  • the length of heavy chain CDR3 regions was average, and there was no unusual occurrence of insertions or deletions.
  • iMOSFLM a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67(Pt 4):271-281.
  • Briney BS Willis JR, Crowe JE. Location and length distribution of somatic hypermutation- associated DNA insertions and deletions reveals regions of antibody structural plasticity.
  • IMGT/junctionanalysis IMGT standardized analysis of the V-J and V-D-J junctions of the rearranged immunoglobulins (IG) and T cell receptors (TR). Cold Spring Harb Protoc. 2011;6: 716-725. doi: 10.1101/pdb.prot5634
  • Herbst-Kralovetz MM et al. (2013) Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells. Emerg Infect Dis 19(3):431- 438.
  • Hutson AM Atmar RL, Marcus DM, Estes MK. Norwalk virus-like particle hemagglutination by binding to h histo-blood group antigens. J Virol. 2003;77: 405-415. doi: 10.1128/JVI.77.1.405-415.2003
  • McLean GR Torres M, Elguezabal N, Nakouzi A, Casadevall A. Isotype can affect the fine specificity of an antibody for a polysaccharide antigen.
  • LIGPLOT a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 8(2): 127- 134.

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Abstract

Des modes de réalisation selon la présente divulgation concernent des méthodes et des compositions à base d'anticorps monoclonaux destinées à traiter ou à prévenir l'infection par un Norovirus, ainsi que des procédés de production d'anticorps. Des modes de réalisation spécifiques concernent des anticorps monoclonaux qui sont des anticorps qui bloquent la liaison des antigènes de groupes sanguins au Norovirus. Des méthodes de traitement, de prévention et de diagnostic faisant appel auxdits anticorps sont en outre décrites, en plus des compositions d'acide nucléique, de polypeptide et autres servant aux procédés de production desdits anticorps.
PCT/US2016/041763 2015-07-10 2016-07-11 Anticorps monoclonaux humains dirigés contre le norovirus humain et découverte d'épitopes Ceased WO2017011394A1 (fr)

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CN109180810A (zh) * 2018-09-27 2019-01-11 国药中生生物技术研究院有限公司 特异性结合诺如病毒gi.1基因型vp1蛋白和/或vlp的抗体及其制备方法和应用
WO2020046857A1 (fr) * 2018-08-27 2020-03-05 Vanderbilt University Anticorps monoclonaux humains neutralisant les norovirus pandémiques gii.4
CN111019908A (zh) * 2019-12-13 2020-04-17 广东省微生物研究所（广东省微生物分析检测中心） 一种从牡蛎中高效提取诺如病毒的样本高通量前处理方法及试剂盒
WO2020236974A1 (fr) 2019-05-21 2020-11-26 University Of Georgia Research Foundation, Inc. Anticorps se liant à la protéine de fusion du métapneumovirus humain et leur utilisation
EP3604540A4 (fr) * 2017-03-31 2020-12-23 The University of Tokyo Anticorps de norovirus
WO2022173689A1 (fr) 2021-02-09 2022-08-18 University Of Georgia Research Foundation, Inc. Anticorps monoclonaux humains dirigés contre des antigènes pneumococciques

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EP3604540A4 (fr) * 2017-03-31 2020-12-23 The University of Tokyo Anticorps de norovirus
US11530254B2 (en) 2017-03-31 2022-12-20 The University Of Tokyo Norovirus antibody
WO2020046857A1 (fr) * 2018-08-27 2020-03-05 Vanderbilt University Anticorps monoclonaux humains neutralisant les norovirus pandémiques gii.4
US20210324050A1 (en) * 2018-08-27 2021-10-21 Vanderbilt University Human monoclonal antibodies that neutralize pandemic gii.4 noroviruses
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CN109180810A (zh) * 2018-09-27 2019-01-11 国药中生生物技术研究院有限公司 特异性结合诺如病毒gi.1基因型vp1蛋白和/或vlp的抗体及其制备方法和应用
CN109180810B (zh) * 2018-09-27 2021-05-07 国药中生生物技术研究院有限公司 特异性结合诺如病毒gi.1基因型vp1蛋白或vlp的抗体及其制备方法和应用
WO2020236974A1 (fr) 2019-05-21 2020-11-26 University Of Georgia Research Foundation, Inc. Anticorps se liant à la protéine de fusion du métapneumovirus humain et leur utilisation
CN111019908A (zh) * 2019-12-13 2020-04-17 广东省微生物研究所（广东省微生物分析检测中心） 一种从牡蛎中高效提取诺如病毒的样本高通量前处理方法及试剂盒
CN111019908B (zh) * 2019-12-13 2022-10-14 广东省微生物研究所（广东省微生物分析检测中心） 一种从牡蛎中高效提取诺如病毒的样本高通量前处理方法及试剂盒
WO2022173689A1 (fr) 2021-02-09 2022-08-18 University Of Georgia Research Foundation, Inc. Anticorps monoclonaux humains dirigés contre des antigènes pneumococciques

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