Pan-GI norovirus monoclonal antibody and its use

Abstract

The invention provides a protein comprising a heavy chain polypeptide, a light chain polypeptide, or both, wherein the heavy chain polypeptide comprises a sequence of amino acids comprising, consisting essentially of, or consisting of SEQ ID NO: 1, 5-7, 18, 22, and / or 28-30, and wherein the light chain polypeptide comprises a sequence of amino acids comprising, consisting essentially of, or consisting of SEQ ID NO: 2, 8-10, 20, 24, and / or 31-33. Also provided are methods and uses for the inventive protein, including in immunotherapy, prophylaxis, diagnosis, detection, marking or isolation, neutralizing Norovirus GI genogroup, and other immunological uses and methods. Also provided are nucleic acids encoding the inventive protein, vectors comprising such nucleic acids, cells and populations comprising such nucleic acids and vectors, and compositions for therapeutic, prophylactic use as well as for neutralization.

Description

Leydig 773047 NIH E-025-2024-0-PC-01 1 PAN-GI NOROVIRUS MONOCLONAL ANTIBODY AND ITS USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 653,691 filed May 30, 2024, which is incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project number ZIA AI0005022 by the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The Government has certain rights in the invention. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 41,816 Bytes XML file named “773047.XML,” dated May 30, 2025. BACKGROUND OF THE INVENTION

[0004] Human noroviruses are very diverse and are classified into 10 genogroups, further subdivided into at least 49 genotypes. Genogroup I (“GI”) is a group of noroviruses that has been known for decades to circulate in the human population. Immunological reagents directed against subgroups / genotypes within Norovirus genogroup GI, such as antibodies specific for GI.1, GI.2, GI.3, GI.4, GI.5, GI.6, and GI.7 (e.g., GI.1-specific antibody 5I2 (Shanker S. et al., PNAS 113(40), E5830:5837 (2016)) and GI-specific antibody 1227 (Lindesmith L.C. et al., Immunity 50(6), 1530-1542 (2019)) have been developed. Such reagents may have the potential to be used in certain aspects of immunotherapy, prophylaxis, diagnostics, as reagents for norovirus detection, marking or isolation, and the like. However, their use is limited because these extant reagents lack reactivity and binding beyond narrow subgroups within NorovirusLeydig 773047 NIH E-025-2024-0-PC-01 2 genogroup GI. Moreover, current neutralization protocols within the field are limited to specific viruses and culture conditions.

[0005] A pan-Norovirus genogroup GI antibody, or antibody-like reagent, which binds multiple, and desirably all, subgroups / genotypes within Norovirus genogroup GI would be highly desirable. Such a broadly reactive antibody specifically targeting Norovirus genogroup GI would extend the arsenal of immunological therapeutic, prophylactic, detection, isolation, and neutralization agents to more comprehensively, yet specifically, target the entire GI genogroup. However, due to the lack of robust in vitro or ex vivo methods to propagate and test the virus, neutralizing antibodies have been difficult to identify. Indeed, only a few norovirus-neutralizing antibodies have been identified: 5I2 is specific to the GI.1 genotype; 10E9, A1431 and nanobody M4 are specific to GII.4; and NORO-32018 recognizes multiple GII viruses. Thus, since the discovery of noroviruses (GI.1 Norwalk) in 1972 until the present invention, no broadly reactive antibody specifically targeting Norovirus genogroup GI has been described. Accordingly, the present invention addresses a long-felt need in the field. BRIEF SUMMARY OF THE INVENTION

[0006] In an aspect, the invention provides an isolated or substantially pure protein comprising a heavy chain polypeptide, a light chain polypeptide, or both heavy and light chain polypeptides, wherein the heavy chain polypeptide comprises a sequence of amino acids comprising, consisting essentially of, or consisting of SEQ ID NO: 1, 5-7, 18, 22, and / or 28-30, and wherein the light chain polypeptide comprises a sequence of amino acids comprising, consisting essentially of, or consisting of SEQ ID NO: 2, 8-10, 20, 24, and / or 31-33. In other aspects, the invention also provides nucleic acids encoding the inventive protein, vectors comprising such nucleic acids, cells and populations comprising such nucleic acids and vectors, and compositions for therapeutic and prophylactic use as well as for detection and neutralization of Noroviruses within genogroup GI.

[0007] The inventive protein represents the first broadly reactive anti-Norovirus antibody or antibody fragment against the entire GI genogroup. The inventive protein represents a promising agent for clinical applications, and the invention thus provides methods and uses for the inventive protein, including in immunotherapy and prophylaxis, as a diagnostic tool forLeydig 773047 NIH E-025-2024-0-PC-01 3 infection, as a reagent for norovirus detection, marking or isolation, as a pan-Norovirus GI genogroup neutralizing agent, and in other immunological uses and methods. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0008] Figure 1 presents the results of experiments demonstrating that disulfide-stabilized VLPs identify a human norovirus-neutralizing antibody with broad GI recognition. Human B cells (CD19+IgD-) bound to GI.1 DS1 probe were single sorted for further monoclonal antibody (mAb) expression and characterization. Panel a, Side scatter (SSC) gating of GI.1 DS1 probe positive live CD19+IgD- B cells in fluorescence-activating cell sorting (FACS). Panel b, Maturation (CD21) and memory (CD27) markers expressed on sorted B cells (live CD19+IgD- GI.1 DS1+). Panel c, Immunoglobulin isotype (IgA or IgG) of the sorted B cells. Panel d, Light chain types of the sorted B cells. Three antibodies, 16D8, 16E10 and 16H2, were successfully isolated. Ig – immunoglobulin. Panels e-i, Antibody binding against different VLPs: GI.1 wild type (WT) (Panel e), GI.1 DS1 (Panel f), GI.2 (Panel g), GI.3 (Panel h) and GI.7 (Panel i) was characterized in enzyme- linked immunosorbent assay (ELISA). Dots represent a mean of two experiments. Panel j, Half maximal effective concentration was extrapolated from mAb or Fab binding to VLP results in e-i. Dots represent a mean of two experiments. Panel k, Affinity of mAbs to P-domains of GI genotypes was measured in Biocore. Dots represent one measurement. NF indicates Not able to Fit. Panels l and n, Neutralization assay of GI.1 Norwalk norovirus in J4FUT2 enteroid monolayer. The virus was pre-incubated with different concentrations (conc.) of either mAb (Panel p) or Fab (Panel q) for 1 h at 37°C before it was added to monolayers. Replication of the virus was compared at 2 and 24 h post-inoculation by measuring genome copies (gc) of the virus per experimental well (w) in reverse transcription-quantitative polymerase chain reaction (RTqPCR). Results summarize three experiments. Panel n, Nonlinear fit of transformed graphs representing GI.1 Norwalk norovirus neutralization by mAb or Fab, where 100 % is complete neutralization and negative value is enhancement. Neutralization percentages were calculated from a percent reduction in viral genome copies relative to the medium control. Dots represent the mean and error bars – the standard deviation of three experiments.1 μg / mL of Fab corresponds to approximately 20 nM.Leydig 773047 NIH E-025-2024-0-PC-01 4

[0009] Figure 2 presents the results of experiments demonstrating that intravenous infusion with 16E10-LS IgG protects animals from GI.1 norovirus infection. Three rhesus macaques were given intravenous infusions of 16E10-LS IgG antibody (20 mg / kg) 5 days before GI.1 norovirus challenge, while four rhesus macaques received only the virus on the challenge day. Panel a, GI.1 norovirus detection in stool collected from inoculated animals. Animals received GI.1 norovirus orally at 1 x 108gc / animal dose. Dotted line indicates viral RNA detection limit at 104gc / g of stool; stool sampling was discontinued after 2 weeks of negative results for each animal. Data points represent a mean of two technical replicates run twice, and the error bars indicate a standard deviation.16E10-1-3, animals that received 16E10-LS IgG infusion, Control-1-4, animals that received only GI.1 norovirus challenge. Grey shading indicates timespan when viral inoculum may be detected in stool. Panels b-e, Shedding data shown in Panel a as analyzed by different parameters. Each data point is a single animal, and the horizontal line indicates a mean. Statistical analyses were run using Wilcoxon test, with the p value shown above each pairing; d, days; AUC, area under the curve; gc, genome copies. Panels f and g, Serum endpoint (EP) titers of IgM (Panel f) and IgG (Panel g) isotype were measured against GI.1 WT virus-like particle in ELISA. High IgG titers in 16E10 group animals (g) are due to 16E10-LS detection. Data points represent a mean of two technical replicates. Data shown in this figure for animal Control-4 is taken from a previously published study.

[0010] Figure 3 graphically illustrates and presents data demonstrating that cryo-EM structure of antibody 16E10 bound to the norovirus GI.1 P domain reveals an unusually large interface. Panel a, Cryo-EM structure of 16E10 Fab bound to the GI.1 P domain dimer at 2.56 Å, displayed in ribbon representation. Heavy and light chains are labeled to the left of the figure, and antibody domains are labeled to the right; CH1 and CK1 domains modeled from canonical structure as their density was insufficient to delineate details. Reconstruction electron density (5σ), shown as mesh. Panel b, Histogram of buried surface area at the interface for all antibody complexes in the SabDab database, which currently encompasses 7,825 antibody complex structures, with only 2 interfaces larger than 16E10. Panel c, Buried surface areas for P domain contacting antibody segments, which comprise contiguous segments proximal or including the Kabat-defined CDRs that are defined in Figure 10). Panel d, Close-up view of 16E10 bound to left side of the displayed P domain in (Panel a) highlighting the CDR H3, with interactingLeydig 773047 NIH E-025-2024-0-PC-01 5 regions on the P domain colored in dark grey. Panel e, 16E10 epitope with recognizing CDRs with surface areas shown in (Panel d).

[0011] Figure 4 graphically illustrates that 16E10 uses a cavity-based mechanism to enable broad recognition. Panel a, Sequence alignment of P domains from norovirus genotypes GI.1-9 (SEQ ID NOs: 34-42, see Table 5), with 16E10 interactive residues denoted by open ovals for backbone interactions, closed ovals for sidechain interactions, and stars for both. Regions interacting with HBGA and bile acid interacting residues are indicated by bars. Arrows demark divergent residues with larger or smaller side chains across the alignment. Panel b, Conservation mapped onto the structure of the GI.1 P domain, shown in side-view, with conserved residues in white and more diverse shaded. Binding footprint of 16E10 is displayed as white outline. Panel c, Overview of the 16E10 – P domain complex, with interfacial cavity displayed as a mesh. Left panel is blow-up of region with dark grey outline. Panel d, Zoom-in on divergent P domain residues (highlighted) with sidechains that interact with 16E10. In the left two panels, cavities are shown in mesh (for GI.1 residues that are small, which allow larger sidechains to fit); in the center and second-right panels in black mesh (for GI.1 residues that are large, with mesh cavity calculated in the presence of the smallest sidechain in the alignment), and in the rightmost panel in mesh (when the divergent residue faces a water channel). Note that Phe100 in the far left figure is outside the cavity and is not expected to clash with extensions of S239 / S240; however, both GI.4 and GI.6 P domains have K239 and reduced binding to 16E10, indicating that while the cavity mechanism reduces steric clashes, it may not fully ameliorate all sequence alterations.

[0012] Figure 5 graphically illustrates and presents data demonstrating that antibody 16E10 blocks HBGA binding through disruption of P domains on virions. Panel a, Ribbon representation of two 16E10 Fv domains bound to a central norovirus P domain dimer, with HBGA and bile acid shown. Panel b, Model of two 16E10 Fv domains binding to a P domain dimer, which has been fit into the cryo-EM density of the GI.1 DS1 VLP shown in (Panel c). Note that the modeled Fv domains clash with neighboring P domains. Panel c, Cryo-EM reconstructions of the GI.1 DS1 VLP alone and bound to 16E10 Fab, solved to 3.03 Å and 2.32 Å resolution, respectively. Line plot indicates electron density versus radial distance for both reconstructions. Panel d, Three representative NS-EM 2D classes are shown for GI.1 DS1 VLPs, alone (far left), bound to 16E10 Fab, to Fab’2 or to IgG (far right). Schematic representingLeydig 773047 NIH E-025-2024-0-PC-01 6 sample contents are shown in the bottom right of each panel colored according to P domain (light grey), S domain (dark grey) and IgG (grey). Panel e, 16E10 blockade of HBGA binding, measured against VLPs of GI.1, GI.2, GI.3 and GI.7. Dotted lines represent HBGA binding to VLP affinity (500 μM). Panel f, Blockade IC50 (BC50) values calculated from (e-i).

[0013] Figure 6 graphically illustrates and presents data demonstrating that 16E10 recognizes a P domain supersite bound by non-neutralizing and neutralization antibodies of variable breadth. Panel a, Binding competition of 16D8, 16E10 and 16H2 against each other and antibodies 5I2 and A1127 on VLPs from four genotypes (GI.1 DS1, GI.2, GI.3, GI.7). Heat maps, summarizing two experiments, denote reduction in binding of a second antibody when the first antibody is pre- bound. Grey / Not applicable denotes not tested as a second antibody, because the highlighted antibody lacks recognition to the specified genotype; this differs from no inhibition where non-competitive binding could occur. Panels b and c, Broadly reactive norovirus antibodies modeled in complex with P domain (left, P domain in grey surface with antibody in ribbon representation), along with the footprint mapped onto the surface of the P domain colored by conservation within (Panel b) GI or (c) GII (right, 90° orientation) Panel d, Angle of approach of each of the antibodies from (Panels b and c), shown in relation to the GI.1 P domain. Panel e, Overlay of footprints from each of the above antibodies define a supersite for broad norovirus reactive antibodies, mapped onto the GI.1 P domain surface, shaded by conservation of GI and GII genogroups. Panel f, Electrostatic potential of the GI.1 P domain, highlighting an electronegative cleft at the P domain dimer interface.

[0014] Figure 7 graphically illustrates and presents data demonstrating that B cells from human PBMCs bind to GI.1 DS1 probe. B cells from human PBMCs bind to GI.1 DS1 probe. Quantitation of stabilized GI.1 DS1 VLP binding to PBMCs isolated from 10 different human subjects. Cells were stained with a panel used for B cell sorting and collected by a flow cytometer. A sample for norovirus-specific monoclonal antibody isolation was selected based on the GI.1 DS1 binding results and sample availability. Monoclonal antibodies (16D8, 16E10 and 16H2) were isolated from Sample 6. Panel a, A concatenated plot shows that CD19+ cells bind to GI.1 DS1 probe across 10 tested subjects. The dotted line indicates threshold for quantifying percentages summarized in Panel b. Panel b, Frequency of B cells that bind to GI.1 DS1 probeLeydig 773047 NIH E-025-2024-0-PC-01 7 across the tested samples. Data summarize one measurement shown in Panel a. Panel c, A plot showing IgG positive B cells that bind to GI.1 DS1 probe in sample ID6.

[0015] Figure 8 presents sensograms of surface plasmon resonance experiments binding GI P domains to indicated immobilized antibodies. KD derived from global fit of the data are shown above each set of plots. Note differences in y-axis heights. “NF” indicates “Not able to be Fit.”

[0016] Figure 9 graphically illustrates and presents data concerning the cryo-EM validation of GI.1 P domain - 16E10 Fab complex. Panel a, Representative micrograph. Panel b, Representative 2D class averages. Panel c, The local resolution map. Panel d, The gold-standard Fourier shell correlation after non-uniform refinement. Panel e, Heatmap showing the orientations of all particles used in the non-uniform refinement. Panel f, Electron density for selected parts of the protein after focused refinement contorted at 7σ in PyMOL.

[0017] Figure 10 presents the sequence and paratope of 16E10. Panel a, Sequence of 16E10 variable domains (SEQ ID NOs: 1 (upper) and 2 (lower)), with highlighted regions corresponding to regions displayed as cartoon in Figure 2, Panel c. Black dots indicate residues that bury 10Å2or greater in the P domain interface. Panel b, Buried surface area of antibody regions, using exact Kabat definitions of CDRs and framework regions.

[0018] Figure 11 graphically illustrates and presents data concerning the cryo-EM validation of GI.1 VLP alone or in complex with 16E10 Fab. Panel a, Representative micrograph of the GI.1 DS1 VLP (data originally previously published). Panel b, Representative 2D class averages. Panel c, The local resolution map. Panel d, The gold-standard Fourier shell correlation after non-uniform refinement. Panel e, Representative micrograph of GI.1 DS1 VLP bound to 16E10 Fab. Panel f, representative D2 class averages. Panel g, The local resolution map. Panel h, The gold-standard Fourier shell correlation after non-uniform refinement.

[0019] Figure 12 presents representative negative stain micrographs of GI.1 VLPs, alone and with A1227 Fab or different formats of 16E10. In micrographs with 16E10 Fab’2 and IgG, intact VLP particles were sparse, and what is shown is a representative single particle; by contrast micrographs with VLP alone or with Fabs showed large numbers of intact VLP particles, and what is shown is a representative field.Leydig 773047 NIH E-025-2024-0-PC-01 8

[0020] Figure 13 graphically illustrates how heavy and light chains from 16E10 and NORO- 320 overlap with each other, and with a subset of nanobodies. Panel a, 16E10 bound to the norovirus P domain, with the heavy chain colored in darker grey, and the light chain in lighter grey. Panel b, 16E10 overlayed with NORO-320, showing similar binding modes on both heavy and light chains. Panel c, Three previously reported nanobody structures which overlap with the binding surface and angle of the 16E10 light chain, as well as the long CDR H3 loop. Panel d, Three previously reported nanobody structures which overlay with the 16E10 heavy chain, but in all cases do not interact with the P domain dimer cleft as with 16E10.

[0021] Figure 14 presents an image illustrating nanobody embodiments. Norovirus P domains shown in dark and light grey with the 16E10 Fv domain bound. The structure of nanobodies that overlap with the 16E10 heavy chain are shown. In all cases, the left-most loop of the nanobody is proximal to the extended CDRH3 of 16E10. DETAILED DESCRIPTION OF THE INVENTION The inventive protein

[0022] In one aspect, the invention provides an isolated or substantially pure protein comprising a heavy chain polypeptide, a light chain polypeptide, or both heavy and light chain polypeptides, wherein the heavy chain polypeptide comprises a sequence of amino acids comprising, consisting essentially of, or consisting of SEQ ID NO: 5, 6, and / or 7, and wherein the light chain polypeptide comprises a sequence of amino acids comprising, consisting essentially of, or consisting of SEQ ID NO: 8, 9, or 10.

[0023] As noted in the Examples below, the sequences of SEQ ID NOs: 5-7 and SEQ ID NOs: 8-10 are within the antigen-recognition moiety of one exemplary embodiment of the inventive protein (16E10), i.e., the virus-interacting regions (“VIRs”), which overlap with or are contained within the compliment determining regions (“CDRs”) (see, e.g., Figure 10, panel a). Thus, in one embodiment, the inventive protein comprises SEQ ID NOs: 5-7 (heavy chain VIRs) and / or SEQ ID NOs: 8-10 (light chain VIRs). A protein comprising SEQ ID NOs: 5-7 and / or SEQ ID NOs: 8-10 can be, for example, an immunoglobulin molecule (Ig), one exemplary embodiment is discussed in the Examples below: a monoclonal antibody (mAb) referred herein as “16E10.” However, the invention is not limited to only exemplary antibody 16E10 butLeydig 773047 NIH E-025-2024-0-PC-01 9 includes variants as discussed below as well as antibodies 16D8 and 16H2, as disclosed herein. Thus, for ease of reference, in places throughout this description, the inventive polypeptide may be referred to as the inventive antibody or polypeptide comprising an antigen-recognition moiety thereof.

[0024] In another aspect, the inventive protein can comprise a sequence of amino acids comprising, consisting essentially or, or consisting of SEQ ID NO: 7 (AKKIDFPFRGGRRYSDSRPYNTGSLDS), which represents the sequence of the long VIR (VIR3) from the heavy chain component of antibody 16E10, the motif also termed “VIRH3.” In one embodiment, the inventive protein comprising, consisting essentially or, or consisting of SEQ ID NO: 7 can be employed as indicated herein alone (e.g., as a “knob” structure, see, for example, Huang et al., Proc Natl Acad Sci U S A.120(39) (2023): e2303455120, the contents of which are incorporated herein in their entirety). Of course, in embodiments, the inventive protein comprising, consisting essentially or, or consisting of SEQ ID NO: 7 can be included within a larger polypeptide chain, such as an immunoglobulin (e.g., a heavy chain). Alternatively or additionally, the inventive protein comprising, consisting essentially or, or consisting of SEQ ID NO: 7 can be added to or conjugated with a scaffolding polypeptide or other moiety, such as an antibody or nanobody, a nanoparticle, or a de novo designed “mini- protein” scaffold. For example, as illustrated in Figure 14, the CDRH3 moiety can be linked to nanobodies that overlap the heavy chain of 16E10.

[0025] The inventive protein can be or comprise an antibody or polypeptide comprising an antigen-recognition moiety thereof (e.g., a CDR or a VIR) and can be monoclonal or polyclonal. The term “monoclonal,” as used herein, refers to antibodies that are produced by a single clone of producing cells (e.g., B-cells) and bind to the same epitope. In contrast, “polyclonal antibodies” refer to a population of antibodies that are produced by different producing cells and bind to different epitopes of the same antigen.

[0026] Certain embodiments of the invention comprise whole immunoglobulins (“Ig”s), such as IgAs (for example, including IgA1 and IgA2), IgDs, IgEs, IgGs (for example, including IgG1, IgG2, IgG3, and IgG4), or IgMs. Certain other embodiments comprise antibody fragments, which are polypeptides comprising antigen-recognition moieties of antibodies, i.e., one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see,Leydig 773047 NIH E-025-2024-0-PC-01 10 generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005), the entire content of which are incorporated herein in its entirety).

[0027] A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” of each of the heavy and light chains of an antibody, which is responsible for antigen binding.

[0028] An antibody fragment desirably comprises, for example, one or more CDRs or VIRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998), the entire contents of each of which are incorporated herein in their entireties), and (v) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH -VL polypeptide chains to generate a dimeric molecule having two functional antigen binding sites. Antibody fragmentsLeydig 773047 NIH E-025-2024-0-PC-01 11 are known in the art and are described in more detail in, e.g., U.S. Patent Application Publication 2009 / 0093024 A1 (the entire contents of which are incorporated herein in its entirety).

[0029] An antigen-binding portion or fragment of the inventive antibody or polypeptide comprising an antigen-recognition moiety thereof can be of any size so long as the portion binds to the Norovirus genogroup GI virion, hypothesized to be the P-domain thereof. In this respect, an antigen binding portion or fragment of the inventive protein desirably comprises one or more CDRs comprising between about 3 and 27 amino acids (e.g., about 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, or a range defined by any two of the foregoing values). This range is only approximate, and one or more of the CDRs or VIRs can include a longer stretch of amino acids (such as up to about 30, or up to about 35 amino acids, or longer).

[0030] Included in the scope of the invention are functional portions of the inventive protein described herein. The term “functional portion,” when used in reference to the inventive protein, refers to any part or fragment of the inventive antibody or polypeptide comprising an antigen- recognition moiety thereof of the invention, which part or fragment retains the biological activity of the inventive protein of which it is a part (the parent antibody or polypeptide comprising an antigen-recognition moiety thereof). Functional portions encompass, for example, those parts of the inventive protein that retain the ability to bind multiple, desirably all, subgroups within the Norovirus GI genogroup, or detect Noroviruses of the GI genogroup, neutralize Noroviruses of the GI genogroup, or treat or prevent infection with Noroviruses of the GI genogroup, to a similar extent, the same extent, or to a higher extent, as the parent antibody or polypeptide.

[0031] A functional portion of the inventive protein can contain additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent antibody or polypeptide. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., bind multiple, desirably all, strains within the Norovirus GI genogroup. More desirably, any such additional amino acids enhance the biological activity of the antibody or polypeptide comprising an antigen-recognition moiety thereof, as compared to the biological activity of the parent protein. For example, while SEQ ID NOs: 1, 2, 18, 20, 22, and 24 represent the heavy and light variable regions of mAbs 16E10, 16D8, and 16H2, respectively, such can be presentLeydig 773047 NIH E-025-2024-0-PC-01 12 within a larger antibody molecule, such as a whole antibody, through the addition of appropriate constant regions. Such constant regions, including “humanizing” constant regions, are known to those of ordinary skill in the art.

[0032] The invention also provides functional variants of the inventive antibody or polypeptide comprising an antigen-recognition moiety thereof. The term “functional variant,” as used herein, refers to a polypeptide or a protein having substantial or significant sequence identity or similarity to the inventive parent protein, which functional variant retains the biological activity of the parent protein (e.g., the antibody or polypeptide comprising an antigen- recognition moiety thereof) of which it is a variant. Functional variants encompass, for example, those variants of the inventive protein described herein (the parent antibody or polypeptide) that retain the ability to bind Norovirus genogroup GI to a similar extent, the same extent, or to a higher extent, as the parent antibody or polypeptide comprising an antigen-recognition moiety thereof. With reference to the non-limiting examples set forth herein, for example, SEQ ID NOs: 1 and 2 present the amino acid sequences of the heavy and light variable domains, respectively, of antibody 16E10, SEQ ID NOs: 5-10 represent the VIRs thereof, and SEQ ID NOs: 28-33 represent the CDRs thereof as determined using the Kabat definition (see also Figure 10(a)). Thus, a functional variant can comprise an amino acid sequence having a sequence identity to any one of the listed sequences (SEQ ID NOs: 1, 2, 5-10, and / or 28-33) of at least 80%, such as at least 90% or at least 95%. Preferably, a functional variant can comprise an amino acid sequence having a sequence identity to any one of the listed sequences (SEQ ID NOs: 1, 2, 5-10, and / or 28-33) of at least 99%.

[0033] In another aspect, the inventive antibody or polypeptide can comprise all or an antigen-binding portion of one or more of SEQ ID NOs: 18, 20, 22, or 24, or a functional variant or functional portion thereof. It will be observed that SEQ ID NOs: 18 and 20 present the amino acid sequences of the heavy and light variable domains, respectively, of antibody 16D8, and SEQ ID NOs: 22 and 24 present the amino acid sequences of the heavy and light variable domains, respectively, of antibody 16H2. As with the sequences relating to antibody 16D10 (i.e., SEQ ID NO: s 1, 2, 5-10, and 28-33), a functional variant of 16D8 or 16H2 can comprise an amino acid sequence having a sequence identity to any one of the listed sequences (SEQ ID NOs: 18, 20, 22, or 24) of at least 80%, such as at least 90% or at least 95%. Preferably, aLeydig 773047 NIH E-025-2024-0-PC-01 13 functional variant can comprise an amino acid sequence having a sequence identity to any one of the listed sequences (18, 20, 22, or 24) of at least 99%.

[0034] A functional variant can, for example, comprise the amino acid sequence of the parent antibody or polypeptide comprising an antigen-recognition moiety thereof with at least one conservative amino acid substitution. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other and, therefore, resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the same amino acid sub-group, for example, lysine for arginine and vice versa such that a positive charge may be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained; serine for threonine and vice versa such that a free -OH can be maintained; and glutamine for asparagine and vice versa such that a free -NH2can be maintained.

[0035] Alternatively or additionally, functional variants can comprise the amino acid sequence of the parent antibody or polypeptide comprising an antigen-recognition moiety thereof with at least one non-conservative amino acid substitution. “Non-conservative mutations” involve amino acid substitutions between distinct groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent antibody or polypeptide.

[0036] The inventive antibody or polypeptide or functional portions and functional variants thereof also can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example,Leydig 773047 NIH E-025-2024-0-PC-01 14 aminocyclohexane carboxylic acid, norleucine, ^-amino n-decanoic acid, homoserine, S- acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, ^-phenylserine ^- hydroxyphenylalanine, phenylglycine, ^-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N’-methyl-lysine, N’,N’-dibenzyl-lysine, 6- hydroxylysine, ornithine, ^-aminocyclopentane carboxylic acid, ^-aminocyclohexane carboxylic acid, ^-aminocycloheptane carboxylic acid, ^-(2-amino-2-norbornane)-carboxylic acid, ^,^- diaminobutyric acid, ^,^-diaminopropionic acid, homophenylalanine, and ^-tert-butylglycine.

[0037] The inventive antibody or polypeptide or functional portions and functional variants thereof can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt, and / or optionally dimerized or polymerized using techniques well known to persons of ordinary skill in the art. In some embodiments, the inventive antibody or polypeptide or functional portions and functional variants thereof can be conjugated (e.g., to drug moieties, detectable moieties such as fluorescent or radioactive tags, etc.). Furthermore, in some embodiments, the inventive antibody or polypeptide or functional portions and functional variants thereof can be incorporated in an assay, such as via conjugation to or immobilization on a substrate (e.g., paper, cellulose, a composite material, and the like, which are commonly employed as substrates in immunological assays), which can facilitate assays such as enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunosorbent spot (ELISPOT) assays, Western hybridization and the like. Conjugation of the payload moiety to the inventive targeted binding agent, antibody, or polypeptide, or functional portions and functional variants thereof to such moieties or substrates can be achieved by any suitable method, which are known to those of ordinary skill in the art.

[0038] The inventive protein (e.g., antibody or polypeptide or functional portions and functional variants thereof) can be made using methods known in the art. For example, the inventive protein can be made using cells expressing nucleic acids encoding the protein (e.g., within vectors), as described herein. Once produced, the inventive protein then can be isolated and / or purified from the producing cells using methods that are well-known in the art. Accordingly, in an aspect, the invention provides a method for producing the inventive protein,Leydig 773047 NIH E-025-2024-0-PC-01 15 comprising culturing a cell as described herein, which has been transfected or infected with a genetic vector or a nucleic acid encoding the protein, under conditions suitable for the cell or population to produce the protein, and then isolating and / or purifying the protein from the cells or culture. Alternatively, the inventive protein can be manufactured using solid-state protein synthesis technology, as the sequences of exemplary proteins according to the invention are known and some are set forth herein. Nucleic acid

[0039] In an aspect, the invention provides a nucleic acid comprising a nucleic acid sequence encoding the inventive protein. “Nucleic acid sequence” is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides. The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to methylated and / or capped polynucleotides. Non-limiting examples of nucleic acid sequences encoding the antibodies or polypeptides and CDRs thereof referenced in Table 5 (relating to antibody 16E10, 18D8, and 16H2) are set forth herein in SEQ ID NOs: 3 & 4, 17 & 19, and 21 & 23 (encoding full heavy and light chain variable domains, respectively of 16E10, 16D8, and 16H2) and 11-16 (encoding 16E10 VIRs, respectively, as indicated in Table 5), and, in certain embodiments, the inventive nucleic acid molecule comprises a sequence of nucleotides consisting of, or consisting essentially of, or comprising SEQ ID NOs: 3, 4, 17, 19, 21, 23, 11-16, or a combination thereof. Particular embodiments include (a) SEQ ID NOs: 3 and 4, (b) SEQ ID NOs: 17 and 19, and (c) SEQ ID NOs: 21 and 23. Of course, due to the degeneracy of the genetic code, a person of ordinary skill will be well able to generate a nucleic acid sequence encoding any of these exemplary antibodies or polypeptides.

[0040] As noted above, included in the scope of the invention are functional portions of the inventive protein. In reference to a nucleic acid sequence encoding the parent protein, a nucleicLeydig 773047 NIH E-025-2024-0-PC-01 16 acid sequence encoding a functional portion of inventive antibody or polypeptide can encode a protein comprising, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the inventive protein. Also as noted above, included in the scope of the invention are functional variants of the inventive protein. In reference to a nucleic acid sequence encoding the inventive protein, a nucleic acid sequence encoding a functional variant thereof can be for example, about 10% identical, about 25% identical, about 30% identical, about 50% identical, about 65% identical, about 80% identical, about 90% identical, about 95% identical, or about 99% identical to the nucleic acid sequence encoding the parent protein.

[0041] The inventive nucleic acid can be used to make the inventive antibody or polypeptide or functional portions and functional variants thereof, for example using methods known in the art. For example, nucleic acid sequences, polypeptides, and proteins can be recombinantly produced using standard recombinant DNA methodology (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994). Further, a synthetically produced nucleic acid sequence, such as encoding the inventive protein, can be isolated and / or purified from a source, such as a transgenic plant, bacterium, insect, or mammal, e.g., a rat, a human, etc. Methods of isolation and purification are well-known in the art. Alternatively, the nucleic acid sequences, such as but not limited to those described herein, can be commercially synthesized. In this respect, the nucleic acid sequence can be synthetic, recombinant, isolated, and / or purified. Genetic vector

[0042] In one aspect, the invention provides a genetic vector comprising a nucleic acid as described above. The vector can be, for example, a plasmid, a cosmid, a viral vector (e.g., retroviral or adenoviral), and the like. Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., supra, and Ausubel et al., supra).

[0043] In addition to the nucleic acid sequence encoding the inventive antibody or polypeptide or functional portions and functional variants thereof, the vector preferably comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (“IRES”), and the like, which provide forLeydig 773047 NIH E-025-2024-0-PC-01 17 the expression of the nucleic acid sequence in a host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol.185, Academic Press, San Diego, Calif. (1990).

[0044] As noted, one type of vector for use in the context of the invention is a viral vector. Representative viral expression vectors include, but are not limited to, the adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from CRUCELL, INC. (Leiden, The Netherlands)), lentivirus-based vectors (e.g., the lentiviral-based pLP1 from LIFE TECHNOLOGIES (Carlsbad, CA)), and retroviral vectors (e.g., the pFB-ERV plus pCFB-EGSH from STRATAGENE (La Jolla, CA)).

[0045] The vector can be constructed by any suitable method, typically by cloning the desired elements into a vector backbone. In the case of a viral vector, the nucleic acid encoding the inventive antibody or polypeptide or functional portions and functional variants thereof, as well as any other exogenous gene(s) or regulatory element(s), preferably is / are inserted into a site or region (insertion region) in the vector that does not affect the viability of the resultant recombinant virus. Such regions can be readily identified by testing segments of virus DNA for regions that allow recombinant formation without seriously affecting virus viability of the recombinant virus and are generally known to those of skill in the art. For example, a thymidine kinase (TK) gene, such as is present in many viruses, can serve as an insertion region.

[0046] The vector comprising a nucleic acid encoding the inventive antibody or polypeptide or functional portions and functional variants thereof can be introduced into a host cell that is capable of expressing the coding sequence, including any suitable prokaryotic or eukaryotic cell. Preferred host cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently.

[0047] The vector can be introduced into a cell by “transfection,” “transformation,” or “transduction.” The terms “transfection,” “transformation,” or “transduction,” as used herein, refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol.7, Gene Transfer and Expression Protocols, Humana Press (1991));Leydig 773047 NIH E-025-2024-0-PC-01 18 DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle- facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Viral vectors can be introduced into host cells by infection, after growth of infectious particles in suitable packaging cells, many of which are commercially available. Cells

[0048] In one aspect of the invention, a cell (e.g., isolated cell) comprising (1) an inventive antibody or polypeptide or functional portions and functional variants thereof, (2) an inventive nucleic acid sequence encoding an inventive antibody or polypeptide or functional portions and functional variants thereof, and / or (3) a vector comprising an inventive nucleic acid molecule also is provided. Typically, the cell is a mammalian cell, such as are typically employed for the production of recombinant proteins. However, a person of ordinary skill in the art can select a suitable cell for transfection with the nucleic acid sequence encoding the inventive antibody or polypeptide or functional portions and functional variants thereof (see, e.g., Schütz et al., STAR Protocols 4(4), 102572 (2023), which is incorporated herein in its entirety), such as Chinese hamster ovary (CHO) cells, baby hamster kidney cells (BHK), HEK cells, and the like, which are often employed for production of biopharmaceuticals. Of course, the cell type can be non- mammalian, such as insect cells employed in a baculovirus transformation system, yeast, bacteria (e.g., E. coli), etc.

[0049] The inventive cell can be made using standard techniques, which are known to those of ordinary skill. In general, a source cell (or population of cells) is obtained and then can be transduced with a vector (i.e., comprising an open reading frame (“ORF”) that encodes the inventive antibody or polypeptide or functional portions and functional variants thereof) to introduce the genetic construct into the cell (or population of cells). Thereafter, the cell is cultured under conditions suitable for expression of the ORF to produce the inventive antibody or polypeptide or functional portions and functional variants thereof within the cell. Typically, the protocol also involves culturing the cell to proliferate it into a population of cells. The population then can be purified, if desired, and then transduced with a vector, such as describedLeydig 773047 NIH E-025-2024-0-PC-01 19 herein, encoding the inventive antibody or polypeptide or functional portions and functional variants thereof.

[0050] Whichever protocol is employed, the inventive cell, of course, can be proliferated to generate a population of like cells (or a heterogenous population comprising the inventive cell). Such a population of cells can include, for example, at least 105cells / ml, such as at least 106cells / ml, such as at least 107cells / ml, such as at least 109cells / ml, or even greater densities, if desired. It will be observed that the inventive cell (or population thereof) can be used in production of the inventive antibody or polypeptide or functional portions and functional variants thereof, in immunological research in vitro or, in some applications, therapeutically. Compositions

[0051] The inventive protein or functional portions and functional variants thereof nucleic acid, vector, or cell can be formulated as a composition (e.g., pharmaceutical preparation, food or beverage additive, etc.) comprising the inventive protein or functional portions and functional variants thereof, nucleic acid, vector, or cell, and a carrier (e.g., a pharmaceutically or physiologically acceptable carrier). Furthermore, the inventive protein or functional portions and functional variants thereof, nucleic acid, vector, cell, or composition of the invention can be used in the methods described herein alone or as part of a pharmaceutical or other type of formulation.

[0052] The composition (e.g., pharmaceutical preparation) can comprise more than one inventive protein or functional portions and functional variants thereof, nucleic acid, vector, or cell of the invention. Vectors and compositions of the invention can further include or can be administered with (concurrently, sequentially, or intermittently with) any other agents or compositions or protocols that are useful for inhibiting, preventing, or treating a disease or clinical condition. For example, a pharmaceutical composition can comprise one or more other pharmaceutically active agents or drugs. Examples of such other pharmaceutically active agents or drugs that may be suitable for use in the pharmaceutical composition include interferon lambda. As another example, the inventive composition additionally can comprise one or more immunostimulatory / regulatory molecules or adjuvants.

[0053] The carrier used in the inventive pharmaceutical composition can be any of those conventionally used and is limited only by physio-chemical considerations, such as solubilityLeydig 773047 NIH E-025-2024-0-PC-01 20 and lack of reactivity with the active compound(s) or cells, and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) or cells and one which has no detrimental side effects or toxicity under the conditions of use.

[0054] The choice of carrier and manner of formulation of the inventive composition will be determined in part by the particular reagent (protein, cell, etc.), or composition thereof of the invention and other active agents or drugs used, as well as by the particular method used to administer the inventive pharmaceutical composition. A variety of suitable formulations of the pharmaceutical composition, thus, can be employed in the inventive compositions, and in carrying out the inventive methods described herein. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, and intraperitoneal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the inventive polypeptides, nucleic acids, vectors, cells, and compositions of the invention are known, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective response than another route.

[0055] Injectable formulations are among those formulations that are preferred in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622- 630 (1986)).

[0056] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The inventive protein, nucleic acid, vector, cell, and composition can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextroseLeydig 773047 NIH E-025-2024-0-PC-01 21 and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0057] Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, and synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0058] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-b-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0059] Preservatives and buffers may be used. To minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (“HLB”) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

[0060] The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, forLeydig 773047 NIH E-025-2024-0-PC-01 22 injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0061] Other compositions according to the invention can take the form of additives, such as for foodstuffs or beverages. In such embodiments, the inventive protein can be formulated to promote stability and included in a food-safe composition, which can be added to food or beverages. Such compositions can be employed, for example, to neutralize Norovirus genogroup GI in ingestible materials, for example to help combat the spread of Norovirus genogroup GI. Uses

[0062] In an aspect, employing the inventive reagents (i.e., the inventive antibody or polypeptide or functional portions and functional variants thereof, nucleic acid, vector, cell, and composition), the invention provides the clinical use of such in treatment and prophylaxis of infections with viruses within Norovirus genogroup GI. In accordance with such uses and methods, such reagent (e.g., the inventive protein, nucleic acid, cell, population, or composition) can be administered to a subject in an amount and in a location effective to provide treatment or prophylaxis.

[0063] As used herein, the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and / or physiologic effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptom attributable to the disease (i.e., Norovirus infection). To this end, the inventive method comprises administering a “therapeutically effective amount” of the composition comprising the host cells expressing the inventive protein, or a vector comprising nucleic acid sequence encoding the inventive protein. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inventive protein to elicit a desired response in the individual.

[0064] Alternatively, the pharmacologic and / or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a Norovirus genogroup GI infection or symptom thereof. In this respect, the inventive method comprises administering a “prophylactically effective amount” of the composition comprising the host cells expressing the inventive protein,Leydig 773047 NIH E-025-2024-0-PC-01 23 or a vector comprising a nucleic acid sequence encoding the protein, to a subject that is at risk of infection with Norovirus of genogroup GI. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of infection or onset of symptoms).

[0065] Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0066] Of course, the inventive therapeutic or prophylactic method can be achieved using a combination of more than one of the inventive reagents (e.g., a “cocktail” containing two or several, or even all, of the agents, such as the native 16E10 antibody in combination with one or more derivatives thereof, as described herein). For such clinical applications, typically, the subject is human, although the method can be employed with subjects of other species subject to infection with viruses within Norovirus genogroup GI, such as non-human primates.

[0067] For administration to subjects, the dosage of the inventive reagent (the inventive antibody or polypeptide or functional portions and functional variants thereof, nucleic acid, vector, cell, or composition) to be administered in the context of the present invention can be any suitable dosage to achieve therapeutic or prophylactic effectiveness. The suitable dosage will be determined by the treating physician or, in the case of non-human subjects (e.g., non-human primates) the veterinarian or laboratory technician in the exercise of professional judgment. However, for human patients, exemplary suitable dosages of the inventive protein (e.g., the inventive antibody or polypeptide or functional portions and functional variants thereof,) can fall in a range of dosages from between 10 mg and 10,000 mg per patient.

[0068] For administration to subjects, the inventive reagent (the inventive antibody or polypeptide or functional portions and functional variants thereof, nucleic acid, vector, cell, or composition) can be administered using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal,Leydig 773047 NIH E-025-2024-0-PC-01 24 buccal, sublingual, or suppository administration. The reagent preferably is formulated so as to be suitable for oral or parenteral administration. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0069] The inventive reagent can be administered as many times as suitable to achieve the prophylactic or therapeutic effect. Thus, as few as one dose can be administered, or the inventive reagent can be administered twice, three times, or more as deemed advisable by the treating physician, veterinarian, or laboratory technician in the exercise of professional judgment. For therapeutic application, the inventive reagent is administered to a subject suffering from infection with Norovirus genogroup GI, and the patient’s condition is monitored to gauge the improvement in the symptoms. For prophylactic administration, the inventive reagent is administered to a subject prior to challenge with Norovirus genogroup GI.

[0070] Particular application of the inventive therapeutic or prophylactic method and use include immunocompromised patients or in populations at heightened risk for Norovirus infection (such as in schools, cruise ships, encampments, housing for the elderly, and the like) or, for example, to pre-treat subjects prior to travel to a region of the world or to a location presenting a risk of Norovirus infection, or for treating immunocompromised subjects, who may be at risk of chronic infection).

[0071] In another aspect, the inventive protein (e.g., the inventive antibody or polypeptide or functional portions and functional variants thereof) can be used as a neutralizing reagent to treat a sample of interest. Thus, the invention provides a method of neutralizing a Norovirus of genogroup GI within such sample and the use of the inventive protein for neutralizing the Norovirus of genogroup GI within such sample. Accordingly, in addition to or alternatively to the pharmaceutical formulation as discussed herein, the inventive protein can be included as an additive or treatment for foodstuffs or beverages or other material that may pose a threat of infection with Norovirus genogroup GI. The addition of pan-anti-Norovirus genogroup GI neutralizing agents, such as the inventive protein, can neutralize Norovirus genogroup GI virions within such treated materials, thereby lessening the risk of infection upon exposure of human or animal subjects to the treated material.Leydig 773047 NIH E-025-2024-0-PC-01 25

[0072] In practice, this method and use of the inventive protein for neutralizing involves treating the sample by introducing the inventive protein into the sample (when the sample is liquid) or applying the inventive protein to the sample surface or interior (when the sample is solid, such as a tissue sample, which can be in vivo or in vitro). Thusly treated, the inventive protein will bind a Norovirus of genogroup GI within the sample, forming a bound complex, which neutralizes the Norovirus of genogroup GI, rendering the Norovirus of genogroup GI non- infective. The amount of the inventive protein to be added to or exposed to the sample to achieve neutralization can vary but generally can be between 0.1-100 µg / mL.

[0073] In yet another aspect, the inventive protein (e.g., the inventive antibody or polypeptide or functional portions and functional variants thereof) can be used as a reagent for detecting Norovirus genogroup GI within a sample of interest, such as for surveillance, diagnostic, and prevention measures. Thus, the invention provides a method of detecting Norovirus genogroup GI in a sample and the use of the inventive protein for detecting Norovirus genogroup GI within such sample. The sample to be assayed in accordance with the inventive method can be any substance suspected of harboring Norovirus genogroup GI, such as a solid, semisolid, or fluid substance (e.g., a paper or other substrate used for swiping surfaces, tissue from an animal (e.g., a human), excrement from an animal, or foodstuff).

[0074] In practice, this detection method and use of the inventive protein comprising exposing the sample to the inventive protein such that the protein binds Norovirus genogroup GI within the sample to form a bound complex (i.e., comprising the inventive protein bound to Norovirus(es) of genogroup GI within the sample). Thereafter, the presence of the complex within the sample can be assayed. Typically, such assays are performed on samples in vitro, but the inventive method can be used for in situ immunoassays as well.

[0075] Detection of the complex can be achieved by methods known in the art, such as via Western blot, ELISA, ELISPOT, immunoprecipitation, and the like. In such uses, the inventive reagent can be employed using standard techniques as a probe to detect the presence of Norovirus genogroup GI in a sample immobilized on a substrate. In a related application, the inventive antibody or protein or functional portions and functional variants thereof can be immobilized on a substrate and serve as a “capture antibody” in a sandwich ELISA. For tissue samples, in situ hybridization can be performed as well.Leydig 773047 NIH E-025-2024-0-PC-01 26

[0076] The use and method of the invention for detection can be employed diagnostically, for example to identify a pathogen causing symptoms in a patient as a virus belonging to Norovirus genogroup GI. In such application, the sample tested can be fluid or tissue or fluid derived from the subject, such as saliva, blood, vomit, fecal material, urine, or other similar sample, which is then assayed ex vivo. Alternatively, the use and method of the invention for detection can be employed in the context of testing substances to assess their risk of infection, e.g., as surveillance or prevention measures to help combat the spread of Norovirus. Thus, the inventive detection method can be used to assess public facilities (e.g., kitchens, bathrooms, and the like), food or beverages (e.g., designed for human consumption), or other substance for the presence of Norovirus genogroup GI to help gauge the risk of Norovirus infection from contact with such substances. In such applications, the sample to be tested comprises fluid or other material, which can be an aliquot of a fluid-of-interest, a portion of a solid sample (e.g., tissue), or can be collected by swabbing the surface of a material-of-interest.

[0077] To facilitate the use of the inventive reagents in assays, the invention provides a test kit. The kit comprises one or more protein (i.e., an antibody or polypeptide or functional portions and functional variants thereof) according to the invention, one or more reagents for performing an immunological test, instructions for using the kit, and preferably all three of these at least. The test kit can be suitable for use in any desired immunological test, such as, for example, ELISA, ELISPOT, immunoprecipitation, and the like. In this respect, for example, the test kit can take the form of a substrate on which one or more of the antibodies or polypeptides or functional portions and functional variants thereof is immobilized, reagents (e.g., for performing a sandwich ELISA or other suitable assay), and instructions for use. Alternatively, the test can comprise a composition incorporating one or more of the antibodies or polypeptides or functional portions and functional variants thereof (e.g., in solution or lyophilized), a suitable substrate for ELISA assays, reagents for performing ELISA assays, and instructions for use. These examples are only illustrative, as the inventive reagents can be employed in any manner typical of monoclonal antibodies in the performance of immunological assays.Leydig 773047 NIH E-025-2024-0-PC-01 27 EXAMPLES

[0078] The following Examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. In brief, the experiments discussed in the Examples demonstrate the isolation of monoclonal antibodies (“16D8,” “16E10,” and “16H2”) from blood of a human donor, which showed broad binding and blocking specificity across the Norovirus genogroup GI. Although neutralization assays in the field are limited to specific viruses and culture conditions, 16E10 demonstrated neutralization towards the Norovirus genogroup GI as supported by the results of blocking experiments. 16E10 was found to possess high binding affinity and to outcompete other antibodies. Structural analysis revealed an exceptionally large epitope (about 1400Å) for the antibody and indicates that the neutralization occurs by sterically destabilizing the P-domain organization on the surface of the virus. Sequences relating to 16E10 were elucidated and are presented herein as well (see Table 5). EXAMPLE 1

[0079] This example demonstrates the isolation and characterization of the inventive protein. In brief, the example describes the use of a disulfide-stabilized virus-like particle (VLP) to isolate a GI-neutralizing antibody, 16E10, with broad recognition and determine cryo-EM structures to understand its breadth. 16E10 was found to bind a variety of GI genotypes and to neutralize the GI.1 virus in an ex vivo assay with nanomolar half-maximal inhibitory concentration. Moreover, 16E10 is able to abrogate or to reduce infection in NHPs.

[0080] The cryo-EM reconstruction of 16E10 with GI.1-P domain dimer at 2.56-Å resolution reveals an exceptionally large binding surface, located at the cleft of the P domain-dimer interface. This site, part of a supersite that overlaps epitopes of previously-identified antibodies of variable breadth and neutralization, was distal from published receptor-binding sites. Also, a cryo-EM reconstruction of GI.1 VLP reveals that 16E10 disrupts P domains on the VLP surface, explaining the ability of 16E10 to blockade receptor binding. While the surface bound by 16E10 appears to be generally conserved, 16E10 nonetheless recognizes sequence-divergent residues, binding to which appears enabled by cavities in the 16E10-binding interface that accommodate sidechains of diverse size. Broad recognition of norovirus can thus utilize interaction with sequence-divergent residues, through a cavity-based mechanism of diversity tolerance.Leydig 773047 NIH E-025-2024-0-PC-01 28 Methods Human subjects

[0081] Monoclonal antibodies were isolated from PBMCs from a healthy donor at the blood bank of the National Institutes of Health (USA) with unknown history of gastroenteritis. The samples were collected under the VRC National Institutes of Health’s protocol VRC 200 (NCT00067054) in compliance with the NIH Institutional Review Board (IRB) approved protocol and procedures. All subjects met protocol eligibility criteria and agreed to participate in the study by signing the NIH IRB approved informed consent. Research studies with these samples were conducted by protecting the rights and privacy of the study participants. All participants provided informed consent in accordance with protocols approved by the respective IRB and the Helsinki Declaration. Production of VLPs

[0082] VLPs were produced by baculovirus expression in Sf9 cells using the BAC-TO-BAC system (THERMOFISHER). VP1 was cloned into the pFastbac1 vector, and after transformation and blue-white screening in DH10bac cells, bacmid was purified from the E. coli. Bacmid was transfected into Sf9 cell using CELLFECTIN II (THERMOFISHER), and the cells allowed to grow for 7 d to produce P0 virus. Sf9 cells were serially infected two additional times to produced P2 virus which was used for VLP expression. Sf9 cells were infected with the P2 baculovirus at a high MOI, and allowed to grow for 4 d. After expression, cells were removed from the supernatant by centrifugation at 4,000 x g for 30 min, and the solution filtered with a 0.22µM sterile filter. Approximately 30 mL of supernatant was added on top of a 3 mL OPTIPREP (SIGMA ALDRICH) cushion and centrifuged at 75,000 x g for 2 h. The majority of the supernatant was removed, leaving behind ~6 mL total of liquid in the tube, and the supernatant and OPTIPREP were mixed. The sample was then spun at 350,000 x g overnight, after which the visible band was collected by piercing the tube with a needle. The purified VLP was then further polished by size exclusion chromatography on a SEPHACRYL S500 column equilibrated in PBS. In the case of disulfide stabilized GI.1 DS1, an additional step of oxidation was carried out by incubating the VLPs with 20 mM diamide for 2 hours at room temperature,Leydig 773047 NIH E-025-2024-0-PC-01 29 followed by a second round of gel filtration on a SUPERDEX S200 column to remove the oxidation reagents. Near complete to complete oxidation of the disulfides was confirmed by lack of entry into an SDS-PAGE gel under non-reducing conditions. Isolation of monoclonal antibodies (flow staining, sorting, RATP-Ig)

[0083] PBMC vials containing approximately 107cells were thawed, washed in PBS and stained with LIVE / DEAD UV BLUE (THERMO SCIENTIFIC) for 20 min at room temperature, followed by incubation for 30 min with the staining cocktail consisting of antibodies: IgD-APC (BD BIOSCIENCE, clone IADB6(IA6-2)), IgA- DyLight405 (JACKSON IMMUNORESEARCH, polyclonal), IgM-BV570 (BIOLEGEND, clone MHM-88), CD21- BV605 (BD BIOSCIENCES, clone B-ly4), CD27-BV650 (BIOLEGEND, clone O323), CD20Ax700 (BIOLEGEND, clone 2H7), CD19-PE-Dazzle594 (BIOLEGEND, clone HIB19), IgG-PE-Cy5 (BD BIOSCIENCES, clone G18-145). After antibody cocktail the probe (GI.1 DS1-PE) and additional antibodies (IgKappa-BV711 (BD BIOSCIENCE, clone G20-193) and IgLambda-BV786 (BD BIOSCIENCE, clone JDC12) were added and incubated for 30 min at room temperature for flow cytometry sorting.

[0084] For rapid assembly, transfection, and production of immunoglobulins (RATP-Ig)30, single-cells were sorted in 96-well plates containing 5 µL of TCL buffer (QIAGEN) with 1% β- mercaptoethanol according to the gating strategy shown in Fig.1a. All cell sorts were performed using a BD FACSARIA II instrument (BD BIOSCIENCES) with BD FACSDIVA Software version 9.5.1 (BD BIOSCIENCES). Frequency of antigen-specific B cells were analyzed using FLOWJO 10.8.1 (BD BIOSCIENCES). Reactivity to VLPs by ELISA

[0085] 96-well MAXISORP nunc-immunoplates (THERMO SCIENTIFIC) were coated with 0.1 µg / ml of protein (VLPs: GI.1 DS119, GI.1 WT, GI.2, GI.3, GI.7, GenBank accession numbers: M87661, AJ277610, AB187514, AJ277609 respectively) in PBS and stored overnight at 4 °C. Plates were washed with PBS / TWEEN20 (0.05%) and blocked with PBS / BSA (3%) for 30 min at 37 °C. Antibodies (16D8, 16E10, 16H2, 5I2, A1227, or VRC01) or Fab (16E10) were serially diluted at 1:5 (8 dilutions per sample) and incubated at room temperature for 1 h in PBS / BSA (0.5%), followed by washing. Antibody detection was carried out by adding mouseLeydig 773047 NIH E-025-2024-0-PC-01 30 anti-human IgG (1:10000; SOUTHERNBIOTECH, clone JDC-10), or, for Fab detection, goat- anti human kappa light chain (1:4000; SOUTHERNBIOTECH) conjugated to horse radish peroxidase (HRP) at room temperature for 1 h, followed by washing. SUREBLUE 3,3’,5,5’Tetramethylbenzidine (TMB) microwell peroxidase substrate (50 µl; KPL) was added at room temperature for 10 min followed by 50 µl 2N sulfuric acid. Data were collected on EPOCH microplate spectrophotometer (BIOTEK) at 450 nm using GEN5 software v.3.10. Reactivity to P domain by surface plasmon resonance

[0086] The binding kinetics of GI P domains to 16E10, 16H2, and 16D8 antibodies were assessed by surface plasmon resonance on a BIACORE T-200 (GE HEALTHCARE) in 1X HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20) buffer at 25 °C. The antibodies were diluted to 2 μg / mL using 2.4 mM sodium acetate solution at pH 5 and were immobilized onto a CM5 chip to approximately 500 RU. P domains were applied to the antibody immobilized chip with serially diluted concentrations ranging from 10 μM to 0.61 nM HBS-EP+ buffer served to account for nonspecific binding and instrument noise. The association phase was carried for 60 s at a 30 μl / min flow rate, followed by a dissociation phase for 30 s at the same flow. The chip was regenerated between experiments using REGENERATION SOLUTION buffer (3M Magnesium chloride) for 60 s. The concentration series were fitted globally with BIACORE T200 evaluation software using a 1:1 model of binding. Binding kinetics plots were generated using GRAPHPAD PRISM 9.0. VLP-carbohydrate mAb blocking assay

[0087] 96-well MAXISORP nunc-immunoplates (THERMO SCIENTIFIC) were coated with a mix of HBGAs (A-1, A-2, B-1, B-2 (ELICITYL), Lea, Lex, Leb, Ley (ISOSEP)) containing 1 µg / mL of each for 2 h at 37 °C prior they left at 4 °C overnight. VLPs at 2 µg / mL (final concentration 1 µg / mL) were pretreated with serially diluted concentrations (1:4) of each mAb or Fab for 1 h at 37 °C. Plates were washed with PBS / TWEEN20 (0.05%) and blocked with PBS / BSA (3%) for 30 min at 37 °C. VLP-mAb / Fab complexes were added to the HBGA- coated and blocked plates and incubated for 1 h at 37 °C in PBS / BSA (0.5%), followed by washing. VLP binding to HBGAs was detected with A1227 mAb (0.5 µg / mL) in PBS / BSA (0.5%) for 1 h at room temperature. After washing the plates, a secondary antibody mouse anti-Leydig 773047 NIH E-025-2024-0-PC-01 31 human IgG (1:10000; SOUTHERNBIOTECH, clone JDC-10) conjugated to horse radish peroxidase (HRP) was added and incubated for 1 h at room temperature, followed by washing. SUREBLUE 3,3’,5,5’- TMB microwell peroxidase substrate (50 µl; KPL) was added at room temperature for 10 min followed by 50 µl 2N sulfuric acid. Data were collected on Epoch microplate spectrophotometer (BIOTEK) at 450 nm using Gen5 software v.3.10. Antibody competition assay

[0088] 96-well MAXISORP nunc-immunoplates (THERMO SCIENTIFIC) were coated with 1 µg / ml of protein (VLPs: GI.1 DS119, GI.2, GI.3, GI.7, GenBank accession numbers: AJ277610, AAC64603, AJ277609 respectively) in PBS and stored overnight at 4 °C. Plates were washed with PBS / TWEEN20 (0.05%) and blocked with PBS / BSA (3%) for 30 min at 37 °C. Antibodies (VRC01, 16D8, 16E10, 16H2, 5I2 and A1227) at 10 µg / mL were added individually and incubated for 30 min at room temperature. Without a wash in between 1 µg / mL of biotinylated monoclonal antibodies (16D8, 16E10, 16H2, 5I2 and A1227) were added to designated wells and incubated for 1 h at room temperature. After washing, STREPTAVIDINHRP (THERMO SCIENTIFIC) was added and incubated for 1 h followed by development of the plates with SUREBLUE 3,3’,5,5’- TMB microwell peroxidase substrate (50 µl; KPL) at room temperature for 10 min and 50 µl of 2N sulfuric acid afterwards. Data were collected on Epoch microplate spectrophotometer (BIOTEK) at 450 nm using GEN5 software v.3.10. Virus neutralization assay

[0089] Human jejunal intestinal enteroids (J4Fut2 HIEs) were kindly provided by Dr. Mary K. Estes’ lab and were plated and differentiated as cell culture monolayers in collagen IV-coated 96-well plates in commercial INTESTICULT human organoid growth medium (INT; STEM CELL TECHNOLOGIES), as previously described. Prior to infection, 5-fold serial dilutions of 16E10 IgG, 16E10 Fab, 5I2 IgG, 5I2 Fab, VRC01 IgG, or VRC01 Fab were prepared in CMGF(−) medium supplemented with 500 μM glycochenodeoxycholic acid (GCDCA; SIGMA, G0759), and each dilution or the medium control was mixed in equal volume with 100 TCID50 of GI.1 norovirus (GI.1-ID7-100595). The antibody fragment:virus mixtures were preincubated for 1 h at 37 °C prior to inoculation onto triplicate wells of the differentiated J4Fut2 HIELeydig 773047 NIH E-025-2024-0-PC-01 32 monolayers and incubated for additional 2 h at 37 °C. After 1 h postinfection (hpi), monolayers were washed twice with CMGF(-) medium and incubated with differentiation INT medium supplemented with 500 µM GCDCA. After 2 hpi (immediately after wash) and 24 hpi, cells and medium were collected, and RNA was extracted using DIRECT-ZOL RNA MINIPREP kit (ZYMORESEARCH) following manufacturer’s protocol. RNA extracted at 2 hpi was used to determine a baseline value for the amount of input virus that remained associated with cells after washing the inoculated cultures. Virus replication was assessed by quantifying virus genome equivalent levels (GEs) from samples extracted at 24 hpi in comparison to the 2 hpi time point. Percent reduction in GEs relative to medium (100%) was determined, and neutralization percentages from a percent reduction in viral genome relative to the medium control were calculated. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed as described previously. Macaques

[0090] The experiment was approved by Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH and complied with ethical regulations at the respective institutions (Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH and of Bioqual, Inc.). Macaques were housed and cared for in accordance with local, state, federal, and institute policies in facilities accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International, under standards established in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Healthy male and female, Indian- (n = 1) and Chinese-origin (n = 6) rhesus macaques (Macaca mulatta) were used in this study. Macaques were monitored for physical health, food consumption, body weight, temperature, complete blood counts, and serum chemistries. Monoclonal antibody infusion

[0091] Rhesus macaques were randomized into mAb infusion group (n =3) and control group (n = 4) based on gender and weight. Macaques received infusions of 16E10-LS mAb (20 mg / kg, 1 ml / min speed) in one leg, the saphenous vein, at BIOQUAL, INC. under sedation five days before the challenge. The monoclonal was stored at -80 °C and thawed before injection.Leydig 773047 NIH E-025-2024-0-PC-01 33 Oral challenge

[0092] Rhesus macaques were sedated and challenged by oral gavage accompanied with sodium bicarbonate (2%) prior and after the administration of the inoculum. We used 1 x 108genome copies per animal of sterile filtered 10% human (GI.1 Norwalk 8WII) that was diluted into sterile water to reach a final volume of 4 ml per inoculum. Viral genome copies per stool preparation were determined by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) using QUANTSTUDIO 6 FLEX thermocycler. Clinical monitoring included regular monitoring of appetite, behavior and activity, weight, complete blood count, serology, consistency of stools, and quantification of viral titer in collected stools. Animals entered biocontainment units following animal biosafety level 2 requirements one month before the challenge and remained in the biocontainment units for another month after demonstrating a virus negative stool sample. Stool processing

[0093] Collected stool was frozen and stored at -80 °C until further processing. On the day of processing, stool was thawed at room temperature. To prepare 10% stool suspensions, 9 ml of sterile PBS was added to 1 g of stool, shook vigorously and vortexed for 1 min twice with 5 min incubation in between at room temperature. The suspension was centrifuged at 1,500 x g for 15 min to remove larger debris from the preparation. The supernatant was collected into a new tube. Unfiltered supernatant was used for viral RNA quantification. Stool suspensions were aliquoted and stored at -80 °C until used. RNA extraction from stool

[0094] Viral RNA was extracted from 10% stool suspensions (100 µl) using QUICK-RNA VIRAL KIT (ZYMORESEARCH) according to manufacturer’s protocol. Samples were stored in 200 µl of DNA / RNA SHIELD (ZYMORESEARCH) and mixed with 600 µl of beta- mercaptoethanol for RNA extraction. During the extraction samples were treated with proteinase K (0.2 mg / ml) and DNAse I (0.125 U / µl). Eluted RNA (25 µl) in nuclease-free water was assessed in RNA quantification.Leydig 773047 NIH E-025-2024-0-PC-01 34 RNA quantification

[0095] RNA quantification was carried out using RT-qPCR. For GI quantification we used primer pair MGBGI_F (CCATGTTCCGYTGGATGC (SEQ ID NO: 25)) / MGBGI_R (GTCHTTMGACGCCATCATCA (SEQ ID NO: 26)) and probe MGBGI_VIC (TGTGGACAGGAGATCGCRATCT (SEQ ID NO: 27)) with TAQMAN FAST VIRUS 1- STEP MASTER MIX (THERMO SCIENTIFIC). Reactions were performed on QUANTSTUDIO 6 Flex thermocycler in a 20 µl reaction volume using the following conditions: 52 °C (10 min), 95 °C (20 s), followed by 45 cycles of 95 °C (15 s) and 60 °C (60 s). A standard curve, generated from 8 consecutive 10-fold dilution of controls, ranging from 103to 1010, based on a recombinant human norovirus DNA for GI and RNA for GII was used to quantitate viral genome equivalents in RNA samples using QUANTSTUDIO REAL-TIME PCR software v1.3. Detection limit for stool samples was 1x104genome copies per g of stool. Serum ELISAs

[0096] IgG and IgM titers to human norovirus VLPs GI.1 WT (GenBank accession number: M87661) were assessed in heat-inactivated serum (56 °C, 30 min).96-well MAXISORP nunc- immunoplates (THERMO SCIENTIFIC) were coated with 0.1 µg / ml of protein in PBS and stored overnight at 4 °C. Plates were washed with PBS / TWEEN20 (0.05%) and blocked with PBS / BSA (3%) for 30 min at 37 °C. Serum diluted at 1:25 was serially diluted at 1:3 (8 dilutions per sample) and incubated at room temperature for 1 h in PBS / BSA (0.5%), followed by washing. Specific antibody isotype detection was carried out by adding goat anti-monkey IgG (1:10000; SOUTHERNBIOTECH), or IgM (1:5000; LSBIO) conjugated to horse radish peroxidase (HRP) at room temperature for 1.5 h, followed by washing. SUREBLUE 3,3’,5,5’- Tetramethylbenzidine (TMB) microwell peroxidase substrate (50 µl; KPL) was added at room temperature for 10 min followed by 50 µl 2N sulfuric acid. Data were collected on Epoch microplate spectrophotometer (BIOTEK) at 450 nm using Gen5 software v3.10 and presented as endpoint titer (reciprocal of last dilution with an OD above the limit of detection) at 0.2, or as 0 where samples did not titer to a cut off of 0.2.Leydig 773047 NIH E-025-2024-0-PC-01 35 Saliva processing

[0097] Animals were sedated and saliva was collected from both sides of the mouth using MICRO-SAL small animal saliva collection kit (OASIS DIAGNOSTICS). The saliva was frozen immediately and stored at 30 °C until processing and the animals were monitored until full recovery from anesthesia. For processing, saliva was thawed at room temperature and boiled at 95 °C for 5 min. The samples were then spun at 13,000 rpm for 5 min, and the supernatant was collected into new tubes. Protein concentration in saliva was measured using NANODROP and the samples were stored at -30 °C until needed. Saliva HBGA phenotyping

[0098] To determine histo-blood group antigen (HBGA) phenotype of macaque saliva, 96- well MAXISORP nunc-immunoplates (THERMO SCIENTIFIC) were coated overnight at 4 °C with 50 µg / ml of saliva in 0.05 M sodium carbonate pH 9.5-9.7 buffer. Plates were washed with PBS / TWEEN20 (0.05%) and blocked with PBS / BSA (3%) for 30 min at 37 °C. We used anti-A (1:100; clone T36; BIOLEGEND), anti-B (1:100; clone CLCP-19B; BIOLEGEND), anti-Lea (1:100; clone T174; BIOLEGEND), anti-Leb (1:100; clone T218 from BIOLEGEND, and clone SPM194 from MYBIOSOURCE), anti-Lex (1:200; clone P12; BIOLEGEND), and anti-Ley (1:100; clone H18A; MYBIOSOURCE) antibodies in PBS / BSA (0.5%) to characterize HBGA phenotype. The antibodies were incubated at room temperature for 1.5 h, followed by washing. Goat anti-mouse Ig polyclonal secondary antibody conjugated to HRP (1:2000; LIFE TECHNOLOGIES) was added to detect specific HBGA at room temperature for 1.5 h, followed by washing. SUREBLUE TMB microwell peroxidase substrate (50 µl; KPL) was added at room temperature for 10 min followed by 50 µl 2N sulfuric acid. Data were collected on Epoch microplate spectrophotometer (BIOTEK) at 450 nm using Gen5 software v3.10. Production of antibodies and P domains

[0099] Antibodies were produced by co-transfection with TURBO293 (SPEED BIOSYSTEMS) of pVRC8400 plasmids coding for the heavy and light chains of the antibody. Twenty-four hours post transfection, ABBOOSTER (ABI SCIENTIFIC) was added to the cells as an expression enhancer. The cells were grown for a total of 5 d, after which the cells were pelleted and the supernatant filtered. MABSELECT SURE (GE) protein A resin was added toLeydig 773047 NIH E-025-2024-0-PC-01 36 the supernatant and then washed with PBS. Antibodies were eluted from the resin with 10 mM glycine, pH 2.0, and immediately neutralized with 100mM Tris, pH 8.0. For the production of Fab or Fab’2 fragments of antibodies, the purified IgG was cleaved with either LysC or pepsin, respectively, back-selected with protein A resin, and polished by size exclusion chromatography on a Superdex S75 column equilibrated in PBS.

[0100] P domains were expressed by transforming E. coli BL21 (DE3) with the P domain construct cloned into the pMAL-c5x vector with an HRV-3C site separating the P domain and MBP tag. The cells were grown at 37 °C. until they reached OD600=0.6, after which the temperature was reduced to 22 °C and protein expression induced with 1 mM IPTG. The cultures were grown overnight, after which the cells were harvested by centrifugation, resuspended in PBS supplemented with DNAse and PMSF, and lysed by sonication. Amylose resin (NEB) was added to the clarified lysate, and the protein washed with PBS, after which HRV-3C protease was added and allowed to cleave overnight. The protein was eluted and further refined by size exclusion chromatography on a SUPERDEX S200 column equilibrated in PBS. Structure determinations

[0101] To determine the structure of 16E10 bound to the GI.1 P domain, we initially tried to crystallize the complex P domain-Fab complex but were unable to obtain suitable crystals. We then proceeded to try single particle cryo-EM by mixing P domain with a 2-fold molar excess of 16E10 Fab, after which the complex was purified by size exclusion chromatography in PBS supplemented with 2 mM β-ME. The complex was concentrated to 2 mg / ml and stored at 4 °C for less than 2 d before freezing grids. Immediately before freezing, the complex was diluted with 50mM sodium acetate pH 5.0, 150 mM NaCl to 0.1 mg / ml. The sample was incubated in 0.085 mM n-Dodecyl β-D-maltoside (DDM) for 10 min and spotted onto CARBON FILM 200 MESH, Cu grids. Complexes were vitrified using a VITROBOT MARK IV with a wait time of 30 s and a blot time of 3 s. Data were acquired using the LEGINON system 42 on a TITAN KRIOS electron microscope operating at 300 kV and fitted with GATAN K3 direct detection device. The dose was fractionated over 50 raw frames and collected over a 2.5 s exposure time.

[0102] For the structure determination of 16E10 bound to the GI.1 VLP, 2.7 µL of the complex at 1 mg / ml was added to a QUANTIFOIL 2x2 gold grid and plunge frozen using a VITROBOT with a blot time of 2.5 s. Data was collected on a at the NICE cryo-EM facilityLeydig 773047 NIH E-025-2024-0-PC-01 37 using a FEI TITAN KRIOS microscope with a GATAN K3 SUMMIT DED detector operated in super-resolution mode (pixel size before binning: 0.415 Å).

[0103] Previously collected cryo-EM images of the GI.1 VLP alone19 were re-processed to have the same analysis as that of the VLP complex. All cryo-EM reconstructions were performed using the CRYOSPARC v4.2.1 package, and structural building and refinements were performed in COOT33 and PHENIX34, respectively. Analysis of the structures was performed using the PDBEPISA SERVER35, and figures were generated using PYMOL, CHIMERA36, ESPRIPT37. Conservation was calculated as normalized Shannon’s entropy. For GI structures conservation was calculated based on 9 genotypes (GI.1-GI.9). For GII structures conservation was calculated based on 27 GII genotypes (GII.1-GII.27). Negative-stain electron microscopy

[0104] Norovirus VLPs or VLP / antibody mixtures were diluted with buffer containing 10 mM HEPES, pH 7, and 150 mM NaCl, adsorbed to glow-discharged carbon-coated copper grids for 15 s, washed three times with the same buffer and negatively stained with 0.7% uranyl formate. Micrographs were collected on a THERMOFISHER TALOS F200C electron microscope operated at 200 kV and equipped with a 4k × 4k Ceta camera at a nominal magnification of 57,000 corresponding to a pixel size of 0.25 nm. Reference-free 2D classification was performed using Relion38. Statistical methods

[0105] To test for differences between NHPs in control and monoclonal antibody infusion groups, we used Wilcoxon test in JMP version 16. Results Human monoclonal antibody recognizing multiple GI genotypes

[0106] For norovirus-specific monoclonal antibody isolation, we used peripheral blood polymorphonuclear cells (PBMCs) collected from a blood donor that was selected based on B cell binding to GI.1 disulfide-stabilized VLPs19 (GI.1 DS1) (Figure 6, panels a, b). The selected blood donor had a reasonably high frequency of GI.1 DS1-specific IgG B cells (Figure 6, panel c). We used the selected blood donor for isolation of monoclonal antibodies specific to norovirusLeydig 773047 NIH E-025-2024-0-PC-01 38 by sorting and sequencing single B cells that bound to a GI.1 DS1 VLP (Fig.1a). Of the 38 that were sorted and sequenced, we selected and expressed three (16D8, 16E10 and 16H2) from mature memory B cells (CD21+CD27+) (Fig.1b). Of these, one was originally of IgA (16D8) and two of IgG (16E10 and 16H2) isotypes (Fig.1c), and all had kappa light chains (Fig.1d); we expressed all as IgGs. All three antibodies derived from different heavy and light chain origin genes.16E10 had the longest heavy chain third complementarity-determining region (CDR H3) (27 amino acids) and the highest frequency of somatic hypermutation (SHM) (12.6% on heavy chain) (Table 1).

[0107] Binding specificity of these three monoclonal antibodies against five VLPs of GI genogroup (GI.1 wild-type (WT), GI.1 DS1, GI.2, GI.3 and GI.7) was measured in an enzyme- linked immunosorbent assay (ELISA) (Fig.1e-i). Coating with different VLPs was controlled by previously characterized antibodies, including 5I2 (specific to GI.114), A1227 (cross-reacts with all GI genotypes 20) and VRC01 (an HIV antibody that does not bind to any of the GI noroviruses). Each of our three identified antibodies bound to GI.1 VLPs. Notably, 16E10 had similar low half maximal effective concentrations (EChms) across all tested VLPs (Fig.1j), which was very little affected by the size of the

[0108] IgG when the same binding was tested with 16E10 fragment antigen-binding region (Fab) fragments (Fig.1j).16D8 exhibited its lowest EChm against GI.3 VLP (Fig.1j), and16H2 had similar EChm against GI.3 as GI.1 WT and DS1 VLPs (Fig.1j). To assess whether specificity of these antibodies was directed to P domains, we measured the P domain affinity for six genotypes of GI genogroup (Fig.1k and Figure 7). Indeed, all the antibodies bound to most of the tested P domains, with 16E10 generally demonstrating the highest affinities across all tested P domains and 16H2 having lower affinity or not binding more isolating P domains than the other antibodies.

[0109] We next examined if the broad activity of 16E10 could also be reflected in neutralization against noroviruses. Only GI.1 Norwalk virus out of GI genogroup noroviruses can efficiently replicate in human enteroids. We preincubated the virus with 16E10, either IgG or Fab, and transferred the complexes onto human enteroid cultures. Both complexes showed inhibition of norovirus replication (Fig.1l-n), where half maximal inhibition concentration was at least 10-fold lower for 16E10 IgG (26.3 µM) when compared to 16E10 Fab (274 µM).Leydig 773047 NIH E-025-2024-0-PC-01 39 Protection against GI.1 norovirus long shedding by 16E10

[0110] To test whether 16E10, which showed promising activity in vitro (Fig.1), might correlate with protection in vivo, we used our norovirus model in non-human primates (NHPs) 23, and included the LS mutation in the 16E10 constant region to enhance mucosal targeting 24. Since only animals with histoblood group antigen phenotype Leb and lacking blood group B are permissive with GI.1 norovirus 23, we screened 147 animals and identified 7 susceptible rhesus macaques (Table 2). We randomized the selected animals into 2 groups. One group of three animals received 16E10-LS intravenously at 20 mg / kg dose and was challenged with GI.1 norovirus orally (1x108genome copies per animal) 5 days after the infusion. Another group of four animals received only GI.1 norovirus challenge. Overall, none of the 16E10-infused animals shed beyond the first 2 days, when inoculum is passing through the animal and may be detected (Fig.2a). No significant difference in the onset of virus shedding was observed (Fig.2b), although infused animals stopped shedding significantly earlier than controls (Fig.2c). In addition, total viral genome copies detected in the stool, area under the curve (AUC), from both groups was lower in the animals that received 16E10-LS infusion (Fig.2d). Of note, the peak viral titers between the two groups were roughly equivalent (Fig.2e). The antibody-infused group did not develop serum responses against the GI.1 VLP, beyond the initial infusion, while the control group demonstrated IgM (Fig.2f) and IgG titers (Fig.2g) by week 2 after challenge. IgG titers in the serum of the infused group, likely infused 16E10-LS (Fig.2g), suggested a ~4 week half-life. Overall, 16E10-LS infused intravenously before a GI.1 norovirus challenge abrogated or reduced infection in NHPs. Structure of 16E10 with GI.1 P domain

[0111] In light of the protective ability of 16E10, we used single particle cryo-EM to determine the structure of the antigen-binding fragment (Fab) of 16E10 in complex with the isolated P domain dimer from the GI.1 Norwalk strain at 2.56-Å resolution (Figure 8; Table 3). Two Fabs bound symmetrically to opposing faces of a single P domain dimer (Fig.3a). If the P domain were positioned in the context with the full virus, each Fab would have an approach angle of ~50° degrees relative to the viral surface (Fig.3b). Only the variable domains (Fv) were well-ordered in the cryo-EM reconstruction, with the CH1 and CK1 domains displaying poor density, likely owing to flexibility in the elbow region of the Fab (Fig.3b). Overall, 16E10 hadLeydig 773047 NIH E-025-2024-0-PC-01 40 an unusually large interface with the P domain, burying 1396 Å2 of solvent accessible surface, which was within the top 0.05% of antibody interface surfaces in the Protein Data Bank (PDB) (Fig.3c).

[0112] The major determinant of the binding derived from the extended CDR H3 of the antibody, which inserted into a cleft between P domain protomers and buried over 800 Å2 at the interface with the P domain (Fig.3d, e). In addition, the CDR H1 and H2 loops also contributed to the interaction, burying ~230 and ~160 Å2, respectively, along with the heavy chain framework region 3 (FR H3) (Figure 9). Most of the light chain did not contact the P domain, though the light chain of 16E10 contributed binding through its CDR L2 loop, interacting with a region of the P domain more distal to the dimer interface than the rest of the antibody (Fig 3f). Overall, the structural analysis revealed 16E10 to bind with a remarkably large surface area, primarily contributed by its extended CDR H3.

[0113] Accordingly, while the CDRs as determined using the Kabat definition are represented with overbars in Figure 10(a), functionally, the virus-interacting regions (“VIRs”), which are proximal to and / or overlap with the CDRs have been determined to be as represented by dots in Figure 10(a) and are represented herein (Table 5) as SEQ ID NOs: 5-10. Cavity-based mechanism of breadth

[0114] Antibodies can overcome diversity by binding to conserved regions on an antigen. However, analysis of the conservation of the 16E10 epitope showed that while the P domain residues proximal to the S domain were largely conserved, portions of the 16E10 binding surface were diverse in sequence (Fig.4a, b). As sequence diversity in limited to sidechains and backbone is conserved, one mechanism that antibodies use to recognize diverse regions is to focus on mainchain atoms; however, we did not observe a focus on mainchain, and sidechains of sequence-diverse residues interacted with 16E10. We did observe, however, that many of the sequence-diverse residues recognized by 16E10 were similar in size and chemistry. Only six residues in the epitope were substantially different in size across GI genotypes. These size discrepancies occurred at P domain residues 239, 240, 251, 310, 318 and 494 (Fig.4a). Notably, each of these residues abutted an interfacial cavity between the P domain dimer and 16E10 or were directed towards solvent (Fig.4c). For Ser239 / Ser240 / Ser251, which have larger side chains in other genotypes, their side chains pointed to cavities, which would allow side chains ofLeydig 773047 NIH E-025-2024-0-PC-01 41 larger size to fit without clash (Fig.4d). In the case of His310 and Ile31, the residues present in the GI.1 genotype were the largest within the genogroup, and thus smaller residues at these positions could be accommodated. Overall, a cavity-based mechanism enables 16E10 to utilize an extraordinarily large interface to recognize divergent noroviruses. 16E10 disrupts P domain organization to block HBGA binding

[0115] The 16E10-P domain complex structure revealed the antibody to bind at a surface distal from that of the reported norovirus ligands, namely HBGAs and bile acid (Fig.5a). However, modeling of the laterally binding 16E10-variable domains into the cryo-EM density of the full norovirus VLP revealed severe clashes with neighboring P domains (Fig.5b). To understand the impact of 16E10 binding, we analyzed the cryo-EM reconstruction of the GI.1 DS1 VLP alone and in complex with Fab (at 3.03 Å and 2.32 Å resolution, respectively) (Figure 10 and Table 3). Remarkably, while density for the P domains in the ligand-free structure was well-defined, in the Fab-bound complex, density in the region corresponding to the P domain had largely disappeared (Fig.5c). By contrast, density for the S domains in both reconstructions was virtually identical, showing that alterations in density were localized to the P domains (Fig.5c). Examination of the radially averaged density of both reconstructions indicated that, in the ligand- free reconstruction, the characteristic lower density at the S domain to P domain hinge at ~155 Å as well as the increased density for the P domain around 200 Å were averaged in the Fab-bound VLP into a plateau or smear from 150-180 Å, with a sharp drop to almost no density by ~200 Å (Fig.5c).

[0116] While steric clashes of a Fab of 16E10 appeared to disrupt the P domains on the surface of the VLP, and as an IgG is substantially larger, we analyzed the impact of Fab’2, and IgG on GI.1 VLPs by negative stain EM. Unexpectedly, both Fab’2 and IgG completely disassembled the disulfide-stabilized VLP (Fig.5d and Figure 11). By contrast, the 16E10 Fab smeared the P domains, but otherwise showed little perturbation of the VLP particle itself.

[0117] Histo-blood group antigens (HBGAs) have been shown to be associated with norovirus susceptibility, enabling glycan-based blocking assays. To interrogate the impact of the P domain perturbations on HBGA binding, we analyzed the blockade ability of 16E10 IgG and Fab on four different genotypes. In specific, we tested the binding of GI.1 WT, GI.1 DS1, GI.2, GI.3 and GI.7 VLPs to HBGA coated plates (Fig.5e-i) and observed both Fab and IgG of 16E10Leydig 773047 NIH E-025-2024-0-PC-01 42 to be capable of blocking VLP interactions with HBGA. In addition, 16E10 demonstrated blocking activity against all tested VLPs. Notably, Fab blocked at lower concentrations than IgG (Fig.5j), suggesting that disruption of the P domains – but not disassembly of the VLP – was responsible for the blockade activity of 16E10. A dimer-cleft supersite with variable vulnerability

[0118] To investigate the prevalence of antibodies recognizing the 16E10 epitope, we used VLP-based competition and tested GI-binding antibodies (Fig.6a).16E10 binding did not reduce the binding of 5I2, a GI-neutralizing antibody known to bind the HBGA-binding site. However, for the remaining tested antibodies which did compete with 16E10, the order of addition made an impact. When 16E10 was the first antibody added, it reduced the binding of the two other isolated GI antibodies reported herein (16D8 and 16H2) as well as the binding of the broadly reactive but non-neutralizing A122716; however, when 16E10 was the second antibody added, no competition was observed to any of the tested antibodies, except with itself. The high affinity of 16E10 for the GI VLPs, with 16E10 able to compete off a lower affinity antibody even if 16E10 were added second may explain this observation.

[0119] Examination of known P domain epitopes showed the GI-reactive antibody A1227 to overlap the binding surface of 16E10, explaining its competition (Fig.6b). In addition, the 16E10 epitope was localized to the same region on the P domain recognized by the broadly GII-reactive antibody NORO320 (Fig.6c), with both heavy and light chains largely overlapping in their approach to the P domain (Figure 12, panels a and b.); moreover, the CDR H3s of both antibodies largely overlapped in binding the dimer cleft. In terms of norovirus nanobodies, the recently reported broadly neutralizing nanobody, M4, overlapped with the 16E10 light chain as well the CDR H3 (Fig.5c), as did Nano-7 and Nano-62. By contrast, nanobodies Nano-30, Nano-53 and Nano-56 overlapped with the 16E10 heavy chain, minus the CDR H3 (Figure 12, panels c and d).

[0120] Despite this multiplicity of overlap, the angles of approach by broadly reactive antibodies showed substantial divergence, ranging from 70° degrees below the horizontal for M4 to 50° degrees above for 16E10 (Fig.6d). Thus, antibodies with largely different approach angles all overlapped to converge on the same supersite (Fig.6e). This electronegative site (Fig.6f) comprised the partially conserved cleft formed at the P domain-dimer interface. Analysis of theLeydig 773047 NIH E-025-2024-0-PC-01 43 antibody characteristics recognizing this supersite revealed a mixture of non-neutralizing and neutralizing antibodies of variable breadth, with 16E10 distinguished by its exceptional buried surface and neutralization breadth (Fig. 6g). Table 1 - immunogenetics of heavy and light chains of GI-specific monoclonal antibodies mAb VH JH CDRH3, CDRH3, VH VK JK CDRL3, CDRL3, VK Isotype # SHM # SHMTable 2 - Rhesus macaques HBGA phenotypesLeydig 773047 NIH E-025-2024-0-PC-01 44 Table 3 - Cryo-EM structure statisticsLeydig 773047 NIH E-025-2024-0-PC-01 45 Table 4 - Broadly reactive antibody characteristicsTable 5 - Sequences

[0121] The sequence of the heavy and light chains of mAb 16E10 were determined and are presented herein as SEQ ID NOs: 1 and 2, respectively. CDRs as determined using the Kabat definition are identified as SEQ ID NOs: 28-33 (see also Figure 10(a)). Virus-InteractingLeydig 773047 NIH E-025-2024-0-PC-01 46 Regions (“VIRs”) within or overlapping with the CDRs are identified as SEQ ID NOs: 5-10. Exemplary nucleic acids encoding the heavy and light chains of mAb 16E10 are identified as SEQ ID NOs: 3, and 4, with the respective VIRs encoded by SEQ ID NOs: 11-16. Amino acid and exemplary coding nucleic acid sequences for mAb 16D8 are presented as SEQ ID NOs: 17- 20. Amino acid and exemplary coding nucleic acid sequences for mAb 16H2 are presented as SEQ ID NOs: 21-24. SEQ ID NO: 1 (16E10 Full Heavy Chain Variable Domain Amino Acid Sequence): VQLLESGGALVHPGGSLRLSCAASGFTFSSSSFSWVRQAPGKGLEWVSGINPSGHDTYY ADSVKGRFTISRDNSKDTLFLEMNSLRAEDTAQYYCAKKIDFPFRGGRRYSDSRPYNTG SLDSWGQGTLVTVSS SEQ ID NO: 2 (16E10 Full Light Chain Variable Domain Amino Acid Sequence): DIVMSQSPVSLPVTPGEPASISCRSSQSLLHSNGYNYVDWYLQKPGQSPQLLLYLGSNRA AGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKLDIK SEQ ID NO: 3 (16E10 Full Heavy Chain Variable Domain Nucleic Acid Sequence): gtgcaattgttggagtctgggggagccttagtacacccgggggggtccctgagactctcctgtgcagcctctggattcacttttagcagtagtt ccttcagttgggtccgccaggctccagggaaggggctggagtgggtctcaggcattaaccctagtggtcatgatacatattacgccgactcc gtgaagggccggttcaccatctccagagacaattccaaggacacactgtttctcgaaatgaacagtctgagagccgaggacacggcccaa tactactgtgcgaagaagatagatttcccatttagaggggggcgacgctactctgatagtcgcccttataatacaggctcccttgactcctggg gccagggcaccctggtcaccgtctcctcag SEQ ID NO: 4 (16E10 Full Light Chain Variable Domain Nucleic Acid Sequence): gatattgtcatgagtcagtctccggtctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgcat agtaatggatacaactatgtggattggtacctacagaagccagggcagtctccacagctcctgttgtatttgggttctaatcgggccgccggg gtccctgacaggttcagtggcagtggatcgggcacagattttacactgaaaatcagcagagtggaggctgaggatgttggggtttattactg catgcaagctctacaaactccctacacttttggccaggggaccaagctggacatcaaac SEQ ID NO: 5 (16E10 VIR1 Heavy Chain Amino Acid Sequence): GFTFSSSSLeydig 773047 NIH E-025-2024-0-PC-01 47 SEQ ID NO: 6 (16E10 VIR2 Heavy Chain Amino Acid Sequence): INPSGHDT SEQ ID NO: 7 (16E10 VIR3 Heavy Chain Amino Acid Sequence): AKKIDFPFRGGRRYSDSRPYNTGSLDS SEQ ID NO: 8 (16E10 VIR1 Light Chain Amino Acid Sequence): QSLLHSNGYNY SEQ ID NO: 9 (16E10 VIR2 Light Chain Amino Acid Sequence): LGS SEQ ID NO: 10 (16E10 VIR3 Light Chain Amino Acid Sequence): MQALQTPYT SEQ ID NO: 11 (16E10 VIR1 Heavy Chain Nucleic Acid Sequence): ggattcacttttagcagtagttcc SEQ ID NO: 12 (16E10 VIR2 Heavy Chain Nucleic Acid Sequence): attaaccctagtggtcatgataca SEQ ID NO: 13 (16E10 VIR3 Heavy Chain Nucleic Acid Sequence): gcgaagaagatagatttcccatttagaggggggcgacgctactctgatagtcgcccttataatacaggctcccttgactcc SEQ ID NO: 14 (16E10 VIR1 Light Chain Nucleic Acid Sequence): cagagcctcctgcatagtaatggatacaactat SEQ ID NO: 15 (16E10 VIR2 Light Chain Nucleic Acid Sequence): ttgggttct SEQ ID NO: 16 (16E10 VIR3 Light Chain Nucleic Acid Sequence): atgcaagctctacaaactccctacactLeydig 773047 NIH E-025-2024-0-PC-01 48 SEQ ID NO: 17 (16D8 Full Heavy Chain Variable Domain Nucleic Acid Sequence): gaggtgaacctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagta tctacaccatgacctgggtccgccaggctccagggaaggggctggagtgggtctcatccattagtagtggtagtacttccatatactacgca gactcagtgaagggccgattcaccgtctccagagacaacgccaagaactcactgtttctgcaaatgaacagcctgagagccgaggacacg gctatatattactgtgcgatatgcggtaggaattataggtccaacaacaactactactacatggaggtctggggcaaagggaccacggtcac cgtctcctcag SEQ ID NO: 18 (16D8 Full Heavy Chain Variable Domain Amino Acid Sequence): EVNLVESGGGLVKPGGSLRLSCAASGFTFSIYTMTWVRQAPGKGLEWVSSISSGSTSIYY ADSVKGRFTVSRDNAKNSLFLQMNSLRAEDTAIYYCAICGRNYRSNNNYYYMEVWGK GTTVTVSS SEQ ID NO: 19 (16D8 Full Light Chain Variable Domain Nucleic Acid Sequence): gaaatagtgatgacgcagtctccaggcaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccagtgagagtgttagca gcaacttagcctggtaccagcagaaacctggccaggctcccaggctcctcattcatggtgcctccaccagggccactggtatcccagccag gttcagtggcagtgggtctgggacagagttcactctcaccatcagcagtctgcagtctgaagattttgcagtttattactgtcagcagtataata actggctcccgatcaccttcggccaagggacacgactggaaattaaac SEQ ID NO: 20 (16D8 Full Light Chain Variable Domain Amino Acid Sequence): EIVMTQSPGTLSVSPGERATLSCRASESVSSNLAWYQQKPGQAPRLLIHGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWLPITFGQGTRLEIK SEQ ID NO: 21 (16H2 Full Heavy Chain Variable Domain Nucleic Acid Sequence): caggaacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatttccggggacagtgtctcta ggagcagtgctacttgggactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacaggtccaagtggttta gtgattatgcattatctgtgaagagtcgaattagcattaacccagacacatccaagaaccagctctccctgcaactgaactctgtgactcccga ggacacggctgtgtattactgtgcaagaggaagaatggggtactactacttcgatctctggggccgtggcaccctggtcactgtctcctcag SEQ ID NO: 22 (16H2 Full Heavy Chain Variable Domain Amino Acid Sequence): QEQLQQSGPGLVKPSQTLSLTCAISGDSVSRSSATWDWIRQSPSRGLEWLGRTYYRSKW FSDYALSVKSRISINPDTSKNQLSLQLNSVTPEDTAVYYCARGRMGYYYFDLWGRGTLV TVSSLeydig 773047 NIH E-025-2024-0-PC-01 49 SEQ ID NO: 23 (16H2 Full Light Chain Variable Domain Nucleic Acid Sequence): gacatccagatgacccagtctccatcctccctgtttgcatctattggagacagagtcaccatcacttgccgggcgagtcagggcattaccaat tccttagcctggtatcagcagaaaccagggacagcccctagcctcctgctgtatgctgcatccagattggaaagaggggtcccatccagatt cagtggcagtggatctgggacggattacactctcaccatcagcggcctgcagcctgaagattttgcaacttattactgtcaacagtattatggt agtccgtacacttttggccaggggaccaagctggagatcaaac SEQ ID NO: 24 (16H2 Full Light Chain Variable Domain Amino Acid Sequence): DIQMTQSPSSLFASIGDRVTITCRASQGITNSLAWYQQKPGTAPSLLLYAASRLERGVPSR FSGSGSGTDYTLTISGLQPEDFATYYCQQYYGSPYTFGQGTKLEIK SEQ ID NO: 25 (MGBGI_F Primer): CCATGTTCCGYTGGATGC SEQ ID NO: 26 (MGBGI_R Primer): GTCHTTMGACGCCATCATCA SEQ ID NO: 27 (MGBGI_R Primer): TGTGGACAGGAGATCGCRATCT SEQ ID NO: 28 (16E10 CDR1 Heavy Chain Amino Acid Sequence): SSSFS SEQ ID NO: 29 (16E10 CDR2 Heavy Chain Amino Acid Sequence): GINPSGHDTYYADSVKG SEQ ID NO: 30 (16E10 CDR3 Heavy Chain Amino Acid Sequence): KIDFPFRGGRRYSDSRPYNTGSLDS SEQ ID NO: 31 (16E10 CDR1 Light Chain Amino Acid Sequence): RSSQSLLHSNGYNYVD SEQ ID NO: 32 (16E10 CDR2 Light Chain Amino Acid Sequence): LGSNRAALeydig 773047 NIH E-025-2024-0-PC-01 50 SEQ ID NO: 33 (16E10 CDR3 Light Chain Amino Acid Sequence): MQALQTPYT SEQ ID NO: 34 (P Domain From Norovirus Genotype GI.1 Amino Acid Sequence): KTRPFTLPNLPLSSLSNSRAPLPISSIGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVA KIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTT PDTFVPHLGSIQANGIGSGNYVGVLSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSV YPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGR NLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGT SEQ ID NO: 35 (P Domain From Norovirus Genotype GI.2 Amino Acid Sequence): KTRAFTVPNIPLQTLSNSRFPSLIQGMILSPDASQVVQFQNGRCLIDGQLLGTTPATSGQL FRVRGKINQGARTLNLTEVDGKPFMAFDSPAPVGFPDFGKCDWHMRISKTPNNTGSGD PMRSVSVQTNVQGFVPHLGSIQFDEVFNHPTGDYIGTIEWISQPSTPPGTDINLWEIPDYG SSLSQAANLAPPVFPPGFGEALVYFVSAFPGPNNRSAPNDVPCLLPQEYITHFVSEQAPT MGDAALLHYVDPDTNRNLGEFKLYPGGYLTCVPNGVGAGPQQLPLNGVFLFVSWVSR FYQLKPVGT SEQ ID NO: 36 (P Domain From Norovirus Genotype GI.3 Amino Acid Sequence): KTKPFSVPNLPLNVLSNSRVPSLIKSMMVSQDHGQMVHFQIGRVTLDGQLQGTTPTSAS QLCKIRGTVYHATGGQGLNLTEIDGTPYHAFESPAPIGFPDLGECDWHINASPANAFTDG SIIHRIDVAQDSTFAPHLGTIHYTNANYDANVSLICSLEWLSPPSGGAPKVNPWAIPRYGS TLTEAAQLAPPIYPPGFGEAIVFFMSDFPIANGSDGLSVPCTIPQEFVTHFVNEQAPTRGE AALLHYVDPDTHRNLGEFKLYPEGFMTCVPNSSGSGPQTLPINGVFTFISWVSRFYQLKP VGTLeydig 773047 NIH E-025-2024-0-PC-01 51 SEQ ID NO: 37 (P Domain From Norovirus Genotype GI.4 Amino Acid Sequence): KTRPFTVPNIPLKYLSNSRIPNPIEGMSLSPDQTQNVQFQNGRCTIDGQPLGTTPVSVSQL CKFRGRITSGQKVLNLTELDGSPFMAFAAPAPAGFPDLGSCDWHIEMSKIPNSSTQSNPI VVNSVKPNSQQFVPHLSSITLDDNVSSGGDYIGTIQWTSPPSDSGGANTNFWKIPDYGSS LAEASQLAPAVYPPGFNEVIVYFMASIPGPNQSGSPNLVPCLLPQEYITHFISEQAPIQGEA ALLHYVDPDTNRNLGEFKLYPGGYLTCVPNSSSTGPQQLPLDGVFVFASWVSRFYQLKP VGT SEQ ID NO: 38 (P Domain From Norovirus Genotype GI.5 Amino Acid Sequence): KTRPFSVPNIPLQLLSNSRVPNLIQSMVLSPDQAQNVQFQNGRCTTDGQLLGTTPVSVSQ ILKFRGKVSAGSKVINLTELDGSPFLAFEAPAPTGFPDLGTSDWHVEMSLNSNSQSSGNPI LLRDIHPNSSEFVPHLGSVCVTAAIEVAGDYTGTIQWTSQPSNVTPVPDVNFWTIPHYGS NLAEASQLAPVVYPPGFGEAIVYFMSPIPGPNTAHKPNLVPCLLPQEFVTHFVSEQAPSM GEAALVHYVDPDTNRNLGEFKLYPEGFITCVPNGTGPQQLPLNGVFVFASWVSRFYQLK PVGT SEQ ID NO: 39 (P Domain From Norovirus Genotype GI.6 Amino Acid Sequence): KTRVFSVPNIPLKDLSNSRVPTLIQGMFVSPDVNQSVQFQNGRCQIDGQLQGTTPVSLSQ LCKIRGRTSSNTRVLNLSEVDGTPFVPLESPAPVGFPDIGGCDWHVGFTFEARDQDPSQN VTFATNDSSFVPYLGSISPHNGDGFHSGDIIGSLDWISAPSDGSALDVWSIPKYGSSLPDV THLAPAVFPPGFGEVILYFHSKFPGSGPTDKLRVPCLIPQEFITHFCNEQAPIAGEAALLHY VDPDTGRNLGEFKLYPDGFMTCVPNSISSGPQTLPINGVFVFVSWVSRFYQLKPVGT SEQ ID NO: 40 (P Domain From Norovirus Genotype GI.7 Amino Acid Sequence): KTRQLTVPNIPLNNLANSRVPAMINKMTVSTDQNQVVQFQNGRCTLEGQLLGTTPVSAS QVARIRGKVFSTASGKGLNLTELDGTPYHAFESPAPLGFPDIGACDWHVFNFQSGSNSFE GSHVQVRYKAKCPICTHLGSIEFTSDQDPAGDQLGTLAWVSPSTSGARVDPWKIPSYGS TVTESTHLAPLIFPPGFGEAIVYFMSDFPIVSGNTAQIPCTLPQEFVSHFVEQQAPIRGEAA LLHYVDPDTHRNLGEFKLYPDGFITCVPNTGGGPQNLPTNGVFVFSSWVSRYYQLKPVG TLeydig 773047 NIH E-025-2024-0-PC-01 52 SEQ ID NO: 41 (P Domain From Norovirus Genotype GI.8 Amino Acid Sequence): RTKPFSVPNIPMNLMSNSRVPMLIDGMMVSNDQNQVPQFQNGRVTLDGQLQGTTTVSA ACIARMRGRIFNNNGNYGVNLAELDGNPYHAFDSPAPLGFPDFGNCDLHMTFVKINPTE LSTGDPSGKVVIHSYDATFAPHLGTVKLEDNNELDQFVGKEVVLELTWVSNRTGATLN LWAVPNYGSNLTQASQLAPPIYPPGFGEAIVYFTSTFPTVSNPKVPCTLPQEFVSHFVNEQ APTRGDAALLHYVDPDTHRNLGEFKMYPEGYMTCVPNAGGGPQTLPINGVFVFISWVS RYYQLKPVGT SEQ ID NO: 42 (P Domain From Norovirus Genotype GI.9 Amino Acid Sequence): KTKQFSVPNLPLNVMSNSRVPSLLNAMVVSPDQAQVVQFQNGRCTLDGQMLGTTTVS ASCVARFRGKTFQAPDNRLGINLAEISGEPYHAFESPAPLGFPDFGDGDWHVTATKVTPS QLEANDPVVVGNVQPYNPQFAPHLGTLVVENPTPDQVATGTDLLFNITWLSNRANNRF NPWVIPNYGSTLTEAAQLAPSIFPPGFGETIVYFNSTFPAVGATTHAAIPCLLPQEFVAHF VNEQAPIRGEAALLHYIDPDTHRNLGEFKIYPEGFVTCVPNVGGTGPQSLPTNGVFVFVS WVSRYYQLKPVGT

[0122] Additionally, sequences for 16D8, 16H2 and 16E10 antibodies have been deposited in GenBank under sequence IDs PP816178, PP816179, PP816180, PP816181, PP816182 and PP816183. The coordinates for the cryo-EM structure of 16E10 bound to the GI.1 P domain has been deposited to the Protein Data Bank with PDB ID 9BOF, and cryo-EM maps of the 16E10-P domain complex, GI.1 VLP and GI.1 VLP bound to 16E10 have been deposited in the EMDB with entry codes EMD- 44734, EMD-44672 and EMD-44673, respectively. These deposits are not an admission that any such information in such deposits is available as of the filing date or constitutes prior art.

[0123] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to beLeydig 773047 NIH E-025-2024-0-PC-01 53 construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0124] The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0125] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 773047 NIH E-025-2024-0-PC-01 54 CLAIM(S):

1. An isolated or substantially pure protein comprising a heavy chain polypeptide, a light chain polypeptide, or both heavy and light chain polypeptides, wherein the heavy chain polypeptide comprises a sequence of amino acids consisting essentially of SEQ ID NO: 5, 6, and / or 7, and wherein the light chain polypeptide comprises a sequence of amino acids consisting essentially of SEQ ID NO: 8, 9, and / or 10.

2. An isolated or substantially pure protein comprising a heavy chain polypeptide, a light chain polypeptide, or both heavy and light chain polypeptides, wherein the heavy chain polypeptide comprises a sequence of amino acids consisting essentially of SEQ ID NO: 28, 29, and / or 30, and wherein the light chain polypeptide comprises a sequence of amino acids consisting essentially of SEQ ID NO: 31, 32, and / or 33.

3. The protein of claim 1 or 2, wherein the heavy chain polypeptide sequence comprises a sequence of amino acids consisting essentially of SEQ ID NO:

1.

4. The protein of any one of claims 1-3, wherein the light chain polypeptide sequence comprises a sequence of amino acids consisting essentially of SEQ ID NO:

2.

5. An isolated or substantially pure protein comprising a sequence of amino acids consisting essentially of AKKIDFPFRGGRRYSDSRPYNTGSLDS (SEQ ID NO: 7).

6. An isolated or substantially pure protein comprising a sequence of amino acids comprising, consisting of, or consisting essentially of SEQ ID NOs: 1, 2, 18, 20, 22, 24 or a combination thereof.

7. The protein of claim 6, wherein the protein comprises sequences of amino acids comprising, consisting of, or consisting essentially of (a) SEQ ID NOs: 1 and 2, (b) SEQ ID NOs: 18 and 20, or (c) SEQ ID NOs: 22 or 24.Leydig 773047 NIH E-025-2024-0-PC-01 55 8. The protein of any one of claims 1-7, which comprises both heavy and light chain polypeptides.

9. The protein of any one of claims 1-8, which is an antibody or antibody fragment that specifically binds to Norovirus GI P-domain.

10. The protein of claim 9, which is an immunoglobulin (Ig).

11. The protein of claim 10, which is an IgG.

12. The protein of claim 10, which is a Fab fragment, a F(ab’)2 fragment, an Fv fragment, an scFv, or a diabody.

13. The protein of claim 5, which is conjugated to a scaffolding polypeptide, antibody, nanobody, or nanoparticle.

14. The protein of any one of claims 1-12, which is conjugated to a detectable moiety and / or a substrate.

15. A nucleic acid molecule comprising a sequence of nucleotides encoding the protein of any one of claims 1-12.

16. A nucleic acid molecule comprising a sequence of nucleotides encoding a protein comprising sequence of amino acids comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or a combination thereof.

17. The nucleic acid molecule of claim 15 or 16, which comprises a sequence of nucleotides comprising SEQ ID NO: 3, SEQ ID NO: 4, 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: 19, SEQ ID NO: 21, SEQ ID NO: 23, or a combination thereof.Leydig 773047 NIH E-025-2024-0-PC-01 56 18. A genetic vector comprising the nucleic acid molecule of any one of claims 15-17.

19. The genetic vector of claim 18, which is a viral vector.

20. A cell comprising the genetic vector of claim 18 or 19.

21. The cell of claim 20, which is a mammalian cell.

22. The cell of claim 21, which is a Chinese Hamster Ovary (CHO) cell or baby hamster kidney (BHK) cell.

23. The cell of any one of claims 20-22, which produces the protein.

24. A population comprising a plurality of cells according to any one of claims 20-23.

25. A composition comprising (a) the protein of any one of claims 1-14, (b) the nucleic acid of any one of claims 15-17, (c) the genetic vector of claim 18 or 19, (d) the cell of any one of claims 20-23, or (e) the population of claim 24, and a pharmaceutically-acceptable carrier.

26. A test kit comprising the protein of any one of claims 1-14, a reagent for performing an immunological test, instructions for use thereof, or a combination thereof.

27. The test kit of claim 26, wherein the immunological test is Western blot, ELISA, ELISPOT, or immunoprecipitation.

28. A method for producing a protein, comprising culturing the cell of any one of claims 20- 23 or the population of claim 24 under conditions suitable for the cell or population to produce the protein and then purifying the protein from the culture.

29. The method of claim 28, wherein the protein comprises the protein of any one of claims 1-12.Leydig 773047 NIH E-025-2024-0-PC-01 57 30. A method of treatment or prophylaxis of Norovirus genogroup GI infection in a human subject in need thereof, comprising administering the composition of claim 25 to a human subject in an amount and in a location effective to provide treatment or prophylaxis of Norovirus genogroup GI infection within the subject.

31. A method for neutralizing Norovirus genogroup GI in a sample, the method comprising treating a sample comprising Norovirus genogroup GI by introducing the protein of any one of claims 1-14 into the sample such that the protein binds the Norovirus genogroup GI within the sample.

32. The method of claim 31, wherein the sample comprises the tissue or fluid of a human subject in vivo.

33. A method of detecting Norovirus genogroup GI in a sample, the method comprising exposing the sample to the protein of any one of claims 1-14 such that the protein binds the Norovirus genogroup GI within the sample to form a complex and detecting the presence of the antibody / Norovirus genogroup GI complex within the sample.

34. The method of claim 33, which is performed by Western blot, ELISA, ELISPOT, or immunoprecipitation 35. The method of any one of claims 31, 33 or 34, wherein the sample comprises a solid, semisolid, or fluid sample ex vivo.

36. The method of claim 35, wherein the sample comprises a tissue or fluid sample obtained from a human subject.

37. The method of claim 36, wherein the sample comprises saliva, blood, vomit, fecal material, or urine.Leydig 773047 NIH E-025-2024-0-PC-01 58 38. The method of any one of claims 31, 33, 34, or 35, wherein the sample comprises food or beverage for human consumption.

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