Nanobody conjugates directed against norovirus

Abstract

The present disclosure generally relates to conjugates, such as fusion proteins of one or more single domain antibodies or nanobodies and one or more immunoglobulin Fc regions, compositions comprising the same and methods of using such conjugates and compositions for the diagnosis, prevention, amelioration and treatment of noroviral infections.

Description

[0001] Nanobody conjugate

[0002] Cross-reference to related applications

[0003] The present application claims priority from Australian Provisional Patent Application No. 2024903243 filed on 8 October 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] Technical field

[0005] The present disclosure generally relates to conjugates, such as fusion proteins of one or more single domain antibodies or nanobodies and one or more immunoglobulin Fc regions, compositions comprising the same and methods of using such conjugates and compositions for the diagnosis, prevention, amelioration and treatment of noro viral infections.

[0006] Background

[0007] Human norovirus was discovered over half a century ago, yet there are still no vaccines, antivirals, or treatments clinically available. These viruses are highly contagious and a major problem in closed institutions such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics.

[0008] Accordingly, there remains an unmet clinical need for effective drug therapies for the prevention and treatment of noroviral infections.

[0009] Summary

[0010] The present disclosure is based on the surprising finding that the conjugation of an Fc region to particular camelid-derived nanobodies (also referred to herein as single domain antibodies) significantly improves their binding affinity across a range of norovirus genotypes. These nanobody conjugates also demonstrated improved neutralization of norovirus in an in vitro model of noroviral infection. Accordingly, the conjugation of these nanobodies to an Fc region of an antibody or immunoglobulin significantly improves their diagnostic design / potential and therapeutic capacities.

[0011] In a first aspect, the present disclosure provides a polypeptide conjugate comprising:

[0012] (a) a single domain antibody that is directed against a P domain of a norovirus, wherein the single domain antibody comprises:

[0013] (i) a CDR1 that comprises the amino acid sequence of RIIFFMYD (SEQ ID NO: 1) or a variant thereof, a CDR2 that comprises the amino acid sequence of QINSDVST (SEQ ID NO: 2) or a variant thereof and a CDR3 that comprises the amino acid sequence of YCNVRRASA (SEQ ID NO: 3) or a variant thereof; or

[0014] (ii) a CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5), GSIFSIYL (SEQ ID NO: 6) or a variant thereof, a CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and a CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8), KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof; and

[0015] (b) an Fc region of an immunoglobulin, or a fragment, variant or derivative thereof.

[0016] Suitably, the single domain antibody comprises the CDR1 that comprises the amino acid sequence of RIIFFMYD (SEQ ID NO: 1) or a variant thereof, the CDR2 that comprises the amino acid sequence of QINSDVST (SEQ ID NO: 2) or a variant thereof and the CDR3 that comprises the amino acid sequence of YCNVRRASA (SEQ ID NO: 3) or a variant thereof. In some examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 4, or a fragment, variant or derivative thereof.

[0017] Suitably, the single domain antibody comprises the CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8) or a variant thereof. In particular examples, the single domain antibody comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 10 or 11, or a fragment, variant or derivative thereof.

[0018] According to particular examples, the Fc region comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 34 or a fragment, variant or derivative thereof. More particularly, the Fc region suitably comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 26 or a fragment, variant or derivative thereof.

[0019] Suitably, the single domain antibody is conjugated, connected or otherwise linked to the Fc region by a hinge region. In particular examples, the hinge region comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 16 or a fragment, variant or derivative thereof.

[0020] Suitably, the polypeptide conjugate comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 15, or a fragment, variant or derivative thereof.

[0021] Referring to various examples, the norovirus is of a GII genogroup, such as of a GII.l genotype, a GII.4 genotype, a GII.8 genotype, a GII.9 genotype, a GII.10 genotype, a GII.14 genotype, a GII.17 genotype, a GII.23 genotype, a GII.24 genotype, a GII.26 genotype, a GII.27 genotype or a GII.NA1 genotype.

[0022] Suitably, the polypeptide conjugate has a KD for the P domain of the norovirus of lower than about 200 nM, lower than about 100 nM, lower than about 70 nM, lower than about 50 nM, lower than about 25 nM, lower than about 10 nM, lower than about 5 nM or lower than about 1 nM.

[0023] In some examples, the single domain antibody has been at least partly humanized.

[0024] For other examples, the polypeptide conjugate further comprises one or more of a detectable marker, a therapeutic agent, a half-life extender and a nanocarrier.

[0025] In a second aspect, the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the polypeptide conjugate of the first aspect.

[0026] Suitably, the isolated nucleic acid comprises, consists of or consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17 to 20, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

[0027] In certain examples, the isolated nucleic acid is or comprises mRNA.

[0028] In a third aspect, the present disclosure provides a genetic construct comprising: (i) the isolated nucleic acid of the second aspect; or (ii) a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector.

[0029] In a fourth aspect, the present disclosure provides a host cell transformed with the nucleic acid molecule of the second aspect or the genetic construct of the third aspect.

[0030] In a fifth aspect, the present disclosure provides a method of producing the polypeptide conjugate of the first aspect, including the steps of: (i) culturing the previously transformed host cell of the fourth aspect; and (ii) isolating the polypeptide conjugate from said host cell cultured in step (i).

[0031] In a sixth aspect, the present disclosure relates to a composition comprising the polypeptide conjugate of the first aspect, the isolated nucleic acid of the second aspect, the genetic construct of the third aspect or the host cell of the fourth aspect and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0032] In a seventh aspect, the present disclosure provides a method of diagnosing or monitoring a norovirus infection and / or a disease, disorder or condition associated therewith in a subject, said method including the step of contacting the subject and / or a biological sample from the subject with the polypeptide conjugate of the first aspect or the composition of the sixth aspect.

[0033] Such a method may further include the step of detecting and / or measuring a level of antigen binding to the polypeptide conjugate. In an eighth aspect, the present disclosure relates to a method of inhibiting or preventing binding of a norovirus to a histo-blood group antigen (HBGA) and / or a bile acid in a subject, said method including the step of administering to the subject a therapeutically effective amount of the polypeptide conjugate of the first aspect, the isolated nucleic acid of the second aspect, the genetic construct of the third aspect, the host cell of the fourth aspect or the composition of the sixth aspect to thereby inhibit or prevent binding of the norovirus to the HBGA and / or the bile acid in the subject.

[0034] In a ninth aspect, the present disclosure provides a method of inactivating or neutralizing a norovirus in a subject, said method including the step of administering to the subject a therapeutically effective amount of the polypeptide conjugate of the first aspect, the isolated nucleic acid of the second aspect, the genetic construct of the third aspect, the host cell of the fourth aspect or the composition of the sixth aspect to thereby inactivate or neutralize the norovirus in the subject.

[0035] In a tenth aspect, the present disclosure provides a method of treating, ameliorating or preventing a norovirus infection and / or a disease, disorder or condition associated therewith in a subject including the step of administering a therapeutically effective amount of the polypeptide conjugate of the first aspect, the isolated nucleic acid of the second aspect, the genetic construct of the third aspect, the host cell of the fourth aspect or the composition of the sixth aspect to thereby treat or prevent the norovirus infection and / or disease, disorder or condition associated therewith in the subject.

[0036] In an eleventh aspect, the present disclosure relates to the use of the polypeptide conjugate of the first aspect, the isolated nucleic acid of the second aspect, the genetic construct of the third aspect, the host cell of the fourth aspect or the composition of the sixth aspect for therapy.

[0037] In a twelfth aspect, the present disclosure relates to the use of the polypeptide conjugate of the first aspect, the isolated nucleic acid of the second aspect, the genetic construct of the third aspect, the host cell of the fourth aspect or the composition of the sixth aspect in the manufacture of a medicament for the treatment and / or prevention of a norovirus infection and / or a disease, disorder or condition associated therewith in a subject.

[0038] In a thirteenth aspect, the present disclosure provides a method for inactivating or neutralising a norovirus associated with a substrate or surface, said method including the step of contacting the substrate with an effective amount of the polypeptide conjugate of the first aspect, or the composition of the sixth aspect to thereby inactivate or neutralise the norovirus associated with the substrate or surface.

[0039] In a fourteenth aspect, the present disclosure provides a kit or device for diagnosing or monitoring a norovirus infection, and / or a disease, disorder or condition associated therewith, in a subject, said kit or device including the polypeptide conjugate of first aspect or the composition of the sixth aspect; optionally one or more reagents for detecting the polypeptide conjugate; and optionally instructions for use.

[0040] Brief description of the drawings

[0041] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood byreference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0042] Figure 1. Structures of novel GII P domains reveals a conserved HBGA pocket. (A) The X- ray crystallography structures of GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1 P domains shown in cartoon representation are mainly equivalent with each other and form a homodimer (i.e., chains A and B). The six P domains were colored, i.e., chain A Pl / chain A P2 / chain B Pl / chain B P2: GII.8 (brown / deepteal / yelloworange / dirtyviolet), GII.14 (hotpink / cyan / green / orange), GII.17 (teal / forest / sand / bluewhite), GII.24 (aquamarine / blue / red / orange / ), GII.26 (yelloworange / purple / smudge / brown), and GII.NA1 (pink / greencyan / palecyan / lightmagenta). (B) Superposition of these six P domains, highlighting Loop A for GII.8 (dirtyviolet), GII.14 (cyan), GII.17 (forest), GII.24 (blue), GII.26 (brown), and GII.NA1 (lightmagenta). A GII.10 P domain A-type complex structure (PDB ID: 3PA1) was superimposed showing the A-type as green sticks. Closeup (right-side) on the conserved residues binding the fucose moiety of HBGAs shown as side-chains or main chains (GII.8 residue numbering). The fucose, galactose, and galactosamine of the HBGA A-type are labeled as Fuc, Gal, and A2G, respectively. (C) Superposition of 15 genotype P domain structures with Loops A, B, C, and D highlighted. The loops were colored as above and with GII.l (yellow), GII.2 (hotpink), GII.3 (lightblue), GII.4 (lime), GII.6 (lightorange), GIL 10 (chocolate), GIL 11 (forest), GIL 12 (salmon), and GIL 19 (deepblue). The GIL 13 (PDB ID: 6JYR) and GII.21 (PDB ID: 4RM0) P domains have unique HBGA binding sites and were omitted from the analysis. The GIL 10 P domain A-type complex structure (PDB ID: 3PA1) was superimposed showing the A-type as green sticks and the HBGA pocket (black circle) surrounded by variable length Loops A and D. Structures are shown from the top and side views. Closeup (right side) highlighting known GII.10 P domain binding residues N355, R356, D385, and G451 within these loops (labeled and shown as sticks). Figure 2. Structure of the engineered GII.4c P domain. (A) The X-ray crystallography structures of the apo GII.4c P domain and H2-type trisaccharide complex are shown in cartoon representation. Only one H2-type molecule bound to the GII.4c P domain dimer and is shown in pale yellow sticks. (B) The GII.4c P domain H2-type complex structure is superimposed with a Gil .4 Saga P domain H2-type complex structure (PDB ID: 4WZK, shown in green sticks) for comparison. The GII.4c amino acid substitutions are shown as side chains on the GII.4c P domain in pink sticks and GII.4 Saga P domain in orange sticks. The closeup (right-side) shows the regular fucose binding residues (labeled and underlined), and amino acid substitutions for GII.4c (pink sticks) and GII.4 Saga (orange sticks). The fucose (Fuc) interacts with GII.4c P domain residues G443, D374, R345, T344, and Y444, whereas S442 interacts with galactose (Gal). (C) Surface representation of GII.4c P domain (top and side views) showing the substituted amino acids and HBGA H2-type superimposed from the GII.4c complex structure. The GII.4c amino acid substitutions are colored cyan and a region of substitutions are circled. Only one set of substitutions are labeled or circled on the dimer for clarity. (D) Surface representation of the GII.4-Syd P domain dimer showing amino acid diversity plot of 2000 different GII.4 sequences. The GII.4 Saga H2- type complex structure (PDB ID: 4WZK) was superimposed to show the HBGA binding site (H2- type, green sticks). The surface color-coded conservation ranged from highly conserved amino acids (purple) to highly variable (white). Predicted highly variable hotspots on the surface of the P domain are labeled and circled and are in comparable regions on the GII.4c P domain. (E) Surface representation of the GIL 10 P domain dimer showing amino acid diversity plot of 29 GII genotype sequences. The GII.4 Saga H2-type complex structure (PDB ID: 4WZK) and GII.10 P domain fucose complex structure (PDB ID: 4Z4R) were superimposed to show the HBGA (H2- type, green sticks) and fucose binding sites (fucose, blue sticks).

[0043] Figure 3. Design and development of Fc-NB26. (A) Amino acid sequence of NB26 (cyan) fused to a human IgG hinge (red) followed by human IgG Fc (green). Fc-NB85 was developed in a similar way. (B) SDS-PAGE of Fc-NB26 (arrow showing Fc-NB26 monomer of 38.4 kDa) and NB26 (arrow showing NB26 of 15 kDa). (C) SEC graph of Fc-NB26 using a Superdex 75 / 200 column (D) SEC of NB26 using a Superdex 75 / 200 column.

[0044] Figure 4. Binding characterization of Fc-NB26 and Fc-NB85 to newly determined norovirus P domain structures. Cross-reactivity of Fc-NB26 and Fc-NB85 to GII.4-CHDC, GII.4c, GII.4- Syd, GII.8, GII.10, GII.14, GII.17-Cs-El, GII.24, GII.26, and GII.NA1 P domains were determined using a direct ELISA with anti-Fc HRP-labeled antibody. Error bars are shown. Figure 5. NB26 and NB85 binding sites are located distant from the HBGA pocket. (A) Superposition of GIL 10 P domain NB26 and NB85 complex (PDB ID: 5004) and the six novel P domain structures (gray cartoons) reveals a conserved NB26 and NB85 binding pocket. NB26 and NB85 binding epitopes on the P domains are colored red and blue, respectively. HBGAs bind to the top of the P domain and are shown as green sticks (B) Closeup of NB26 and NB85 binding site showing GII.10 P domain binding residues and matching GII.8, GII.14, GII.17-Cs-El, GII.24, GII.26, and GII.NA1 P domain residues that could interact with NB26 or NB85. (C) NB26 and Fc-NB26 neutralization was determined using a GII.4 stool sample. Isotype IgG was used as a negative control (-ve ctr). SEM error bars are shown. Significance relative to the negative control was determined using an unpaired T-test to calculate the p values for NB26 (**** p = <0.0001 and * p = 0.0120) and Fc-NB26 (**** p = <0.0001 and * p = 0.0171), and ns = not significant. (D) Norovirus replication curves for NB26 and Fc-NB26. These ICso values were calculated from this analysis is an approximate, since only one concentration less than 50% inhibition is calculated, whereas two below and two above 50% inhibition should be measured for an accurate calculation (I). Error bars are shown. (E) Negative stain electron microscopy micrographs of GII.10 VLPs untreated, treated with NB26, and treated with Fc-NB26. One representative micrograph is shown for each condition. The black arrow shows particle aggregation, and the white arrows show particle disassembly and aggregation. Scale bar represents 200 nm.

[0045] Figure 6. Thermodynamic properties of NB26 and Fc-NB26 binding to P domains using ITC. NB26 or Fc-NB26 was titrated into (A) GII.10 P domain or (B) GII.4-Syd P domain. Examples of the titration (upper) of NB26 or Fc-NB26 to P domains are shown. The binding isotherm was calculated using a single binding site model (lower). (C) Constants K (binding constant in M'1), dl l (heat change in cal / mol), dS (entropy change in cal / mol / deg), and dG (change in free energy in cal / mol) are shown with standard deviations. Fc-NB26 has significantly higher affinity (>100 fold higher) to these P domains than NB26 (Student’s T- test, P value < 0.05).

[0046] Figure 7. Amino acid alignment of 15 GII genotypes used in this study. The Pl-1, P2, and Pl- 2 subdomains are marked with a solid bar (in green for Pl and blue for P2). Highly conserved residues are indicated with an The NB26 (red shade) and NB85 (blue shade) binding epitopes, based on GII.10 P domain NB26 / NB85 complex (PDB ID: 5004), was predicted for the other genotypes using PyMOL and PDBePISA online server.

[0047] Figure 8. Nucleotide and amino add sequences of Fc-NB85. Figure 9. Nucleotide and amino acid sequences of Fc-NB26.

[0048] Figure 10. Four human and one bat norovirus X-ray crystal structures and a corresponding P domain sequence alignment. (A) The P domains of GII.9, GII.23, GII.27, GVIII, and GX are shown in cartoon representation. The asterisks indicated missing residues in the structure, likely due to the flexibility of the loop. The P domain, subdivided into Pl and P2 subdomains, is coloured chain A Pl / chain B Pl and chain A P2 / chain B P2 for GII.9 (sand / light blue and deep purple / split pea), GII.23 (purple / brown and salmon / light blue), GII.27 (lime green / firebrick and deep blue / teal), GVIII (olive / smudge and pink / aquamarine), and GX (hot pink / pale cyan and green / orange), respectively. Loop A is located near the HBGA pocket and commonly contains the essential fucose moiety binding residue (e.g., GII.4 R345) and is longer in GII.23 (~17 residues) and GII.27 (~17 residues) than in GII.9 (~9 residues), GVIII (~9 residues), and GX (~8 residues).

[0049] (B) Clustal W sequence alignment showing the partial P domains of GII.9, GII.23, GII.27, GVIII, GX, and with consensus GII.4 (Syd-2012, JX459908). The P2 subdomains contain numerous insertions and deletions compared to the Pl subdomains. Loop A (pink highlight) and two regular HBGA binding residues in GII.4 P domains (R345 and D374, gray highlight, and termed FUC) are shown. The two equivalent residues in the GII.9 (R346 and D374), GII.23 (R355 and D384), and GII.27 (R355 and D384) P domain structures are positioned similarly to the GII.4 P domain. The GVIII and GX P domains lack these two residues, except for GX D367 (highlighted red), which is located on the equivalent loop and near GII.4 D374. Coordinates and structure factors were depositions into the Protein Data Bank (GII.9-VA97207, PDB ID 9EDM; GII.23- Loretol847, 9EDN; GII.27-Loreto0959, 9EDO; GVIII-Chiba040502, 9EDP; and GX-NPIH26, 9EDQ).

[0050] Figure 11. Fc-NB26 cross-reactivity and epitope binding characterization among these five P domains. (A) Cross-reactivity of Fc-NB26 to GII.9, GII.23, GII.27, GVIII, and GX P domains using direct ELISA with serial diluted Fc-NB26 from a starting dilution of 10 pg / mL in PBS. Error bars are shown (triplicate wells), and the dashed line represents the cutoff at an optical density of 450 (OD450) = 0.05 (15—17). (B) Superposition of GII.10 P domain NB26 complex (PDB ID: 5004) and the five P domain structures (chain A and B coloured light and dark grey, respectively).

[0051] (C) Closeup of equivalent P domain residues (green) potentially interacting with Fc-NB26, where underlined residues show substitutions in GII.9 (chain A: D269, E271, L272, G274, T276, Y459, Q460, E461, T464, and chain B: V231 and P477), GII.23 (chain A: D269, E271, L272, G274, T276, Y459, Q460, E461, P470, and chain B: 1231 and P483), GII.27 (chain A: D269, E271, L272, G274, T276, Y459, Q460, E461, P470, and chain B: 1231 and P483), and with reference GIL 10 (chain A: D269, E271, L272, G274, T276, Y470, Q471, E472, P475, and chain B: 1231 and P488). The residue numbering refers to GIL 10 P domain and the side chains (yellow) interacting with NB26. (D) Closeup of equivalent GVIII P domain residues (green) potentially interacting with Fc- NB26 (chain A: D269, E271, L272, G274, T276, Y476, E477, Q478, P481, and chain B: R231 and A494). (E) Closeup of GX P domain side chains (green) at the equivalent Fc-NB26 pocket shows numerous amino acid substitutions (underlined) that could restrict Fc-NB26 binding (chain A: E270, E272, L273, G275, T277, L455, T456, H457, T460, and chain B: N232 and 0473). Key to the Sequence Listing

[0052] Detailed description

[0053] General Techniques and Definitions

[0054] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0055] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0056] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0057] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0058] Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.

[0059] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0060] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0061] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0062] Throughout the present specification, various aspects and components of the disclosure can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5.5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0063] As used herein, the term “about”, unless stated to the contrary, refers to + / - 10%, more particularly + / -5%, even more particularly + / -1%, of the designated value. The extent of such tolerances and variances are well understood by persons skilled in the art. Typically, such tolerances and variances do not compromise the structure, function and / or implementation of the compositions and methods described herein.

[0064] Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0065] By “consisting essentially of’ in the context of an amino acid sequence, such as a VHH chain or CDR sequence, is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- or C-terminus thereof. By “consisting essentially of’ in the context of a nucleotide sequence is meant the recited nucleotide sequence together with an additional one, two or three amino nucleic acids at the 5’ or 3’ end thereof.

[0066] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference. For the present disclosure, the database accession number or unique identifier provided herein for a gene or protein, as well as the gene and / or protein sequence or sequences associated therewith, are incorporated by reference herein.

[0067] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0068] Single domain antibody conjugates

[0069] The inventors have surprisingly shown for the first time that particular single domain antibodies (also referred to herein as nanobodies) when conjugated to an Fc region or domain surprisingly demonstrate significantly improved binding affinity and neutralization potency against norovirus infection in a cell-culture model. Without being bound by any theory, it is believed that the Fc portion facilitates self-assembly of the single domain antibody conjugates into, for example, dimers. The Fc domain or Fc region may also be useful for extending the half-life of the single domain antibody, or otherwise to provide a desired functionality conferred by the Fc domain or Fc region, such as recognition by a secondary reagent or binding to a solid support for purification.

[0070] Accordingly, the present disclosure provides a polypeptide conjugate that comprises a single domain antibody that is directed against a P domain of a norovirus and an Fc region.

[0071] Suitably, the single domain antibody is nano-85 described in W02016059113, which is incorporated by reference in its entirety herein. As such, in certain examples, the single domain antibody specifically binds to the amino acid sequence of W-V-N-X^F-Y-X2of a norovirus polypeptide, and more particularly a VP1 polypeptide, wherein X1represents any amino acid, preferably Q or P, and X2represents any amino acid, preferably T or S .

[0072] In other broad examples, the single domain antibody is nano-26 described in WO2019057755, which is incorporated by reference in its entirety herein. Accordingly, in such examples, the single domain antibody suitably specifically binds to the amino acid sequence of A- X-A-H-X-H-X-0 of a norovirus polypeptide, and more particularly a VP1 polypeptide, wherein "X" represents any amino acid; "A" represents glutamic acid (E) or aspartic acid (D); "H" represents glycine (G), alanine (A), valine (V), leucine (L) or isoleucine (I), and "O" represents serine (S) or threonine (T). More particularly, the single domain antibody specifically binds to the amino acid sequence of D-X-E-L-X-G-X-T of a norovirus polypeptide, and more particularly a VP1 polypeptide, wherein "X" represents any amino acid. The single domain antibody can further specifically bind to a further epitope on the norovirus polypeptide, such as the amino acid sequence of (N or Q)-(D or E)-(X)is-P with "X" being any amino acid or more particularly Y-Q-E-S-x-P- (X)i2-P with "X" being any amino acid.

[0073] In a particular form, the present disclosure provides a polypeptide or protein conjugate comprising:

[0074] (a) a single domain antibody that is directed against a VP1 polypeptide of a norovirus, such as a P domain thereof, wherein the single domain antibody comprises:

[0075] (i) a CDR1 that comprises the amino acid sequence of RIIFFMYD (SEQ ID NO: 1) or a variant thereof, a CDR2 that comprises the amino acid sequence of QINSDVST (SEQ ID NO: 2) or a variant thereof and a CDR3 that comprises the amino acid sequence of YCNVRRAS A (SEQ ID NO: 3) or a variant thereof; or

[0076] (ii) a CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5), GSIFSIYL (SEQ ID NO: 6) or a variant thereof, a CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and a CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8), KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof; and

[0077] (b) an Fc region of an immunoglobulin, or a fragment, variant or derivative thereof.

[0078] The term “conjugate” or “conjugated” can be used in the context of the present disclosure to describe single domain antibodies disclosed herein that are covalently attached or conjugated to another compound or structure, and more particularly an immunoglobulin Fc region. Accordingly, in one example, the single domain antibodies of the present disclosure are considered to be “conjugated” directly or indirectly to an Fc region of an immunoglobulin, or a fragment, variant or derivative thereof. The polypeptide conjugate may also be referred to or described as a fusion protein / polypeptide or an immunoconjugate. As used herein, the term “immunoconjugate” refers to a polypeptide molecule that includes at least one functional moiety and an antigen binding moiety, such as a single domain antibody disclosed herein.

[0079] In the context of the present disclosure, the terms “single domain antibody”, “VHH”, “VHH antibody fragment”, “VHH chain”, “heavy-chain antibody” and “nanobody” can be used interchangeably herein and denote the variable domain or region of the single heavy chain of antibodies of the type of those found in camelids, which are naturally devoid of light chains. It is noted that the terms “nanobody” and “nanobodies” are registered trademarks of Ablynx N.V. and thus may also be referred to as Nanobody® and / or Nanobodies®.

[0080] In the absence of a light chain, single domain antibodies generally each have three CDRs, denoted CDR1, CDR2 and CDR3 respectively. Additionally, single domain antibodies typically include three or four framework regions (FRs; FR1, FR2, FR3 and optionally FR4). The single domain antibodies described herein can be derived from camel, dromedary, llama or alpaca heavychain antibodies (HCAbs). In particular examples, the single domain antibodies according to the present disclosure are derived from alpaca HCAbs.

[0081] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody (e.g., the VHH chain of a camelid-derived antibody) as defined herein that specifically binds to an antigen and, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. As used herein, the term “complementarity determining regions” (i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region (e.g., a VHH chain) the presence of which are major contributors to specific antigen binding. Each VHH chain of a camelid-derived antibody typically has three CDR regions identified as CDR1, CDR2 and CDR3. “Framework regions” are those variable domain residues other than the CDR residues.

[0082] There are multiple conventions to define, annotate and describe the CDRs (and by extension FRs) of an immunoglobulin or antibody, such as a VHH chain or single domain antibody. To this end, the length and sequence of specific CDRs of an antibody can vary depending upon the specific nomenclature, algorithm or the like used to define them. Exemplary conventions to define CDRs include the Kabat definition (which is based on sequence variability and is the most commonly used; See, e.g., Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991)), the Chothia definition (which is based on the location of the structural loop regions; See, e.g., Chothia, et al., (1987) J Mol. Biol. 196:901-917), the AbM definition (which is a compromise between the Kabat and Chothia definitions and is based on Oxford Molecular's AbM antibody modelling software), the IMGT definition (see, e.g., https: / / www.imgt.org / IMGTindex / CDR.php) and the method described by Kontermann and Diibel (Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). In particular examples, the amino acid sequences of the CDR1, CDR2 and CDR3 of the single domain antibodies of the present disclosure are determined or defined by the Kabat definition. In other examples, the amino acid sequences of the CDR1, CDR2 and CDR3 of the single domain antibodies of the present disclosure are determined or defined by the Chothia definition. In some examples, the amino acid sequences of the CDR1, CDR2 and CDR3 of the single domain antibodies of the present disclosure are determined or defined by a crystal structure thereof. In certain examples, the amino acid sequences of the CDR1, CDR2 and CDR3 of the single domain antibodies of the present disclosure are determined or defined by a 3D structure thereof. In various examples, the amino acid sequences of the CDR1, CDR2 and CDR3 of the single domain antibodies of the present disclosure are determined or defined by binding to a P domain of a VP1 protein of a norovirus.

[0083] In view of the above, the single domain antibody or the polypeptide conjugate provided herein can specifically or selectively bind to a P domain of a VP1 protein of a norovirus. The terms “specifically binds” or “selectively binds” can be used interchangeably herein and shall be taken to mean that the binding interaction between a binding agent disclosed herein (e.g., a single domain antibody of a polypeptide conjugate) and a target molecule described herein (e.g., VP1 protein, such as those set forth in SEQ ID NOs: 35 to 37) is dependent on detection of the target molecule by the binding agent. Accordingly, the binding molecule preferentially binds to or recognizes the target molecule even when present in a mixture of other molecules or organisms. The formation of a complex with a target molecule (e.g., a VP1 protein) that is relatively stable under physiologic conditions can also be a characteristic of such selective or specific binding by a VHH chain of a single domain antibody or a polypeptide conjugate.

[0084] As used herein, the term “binds” refers to the interaction (e.g., the formation of hydrogen bonds) of a binding agent, such as a single domain antibody or a polypeptide conjugate, with a target molecule (e.g., a VP1 or capsid protein) and means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target molecule. For example, a single domain antibody or a polypeptide conjugate of the present disclosure recognizes and binds to a specific structural element (e.g., a P domain) of a VP1 protein of a norovirus rather than to molecules generally.

[0085] Specific or selective binding can be characterized by a KD of about 5xlO-2M or less (e.g., less than 5xlO-2M, less than 10-2M, less than 5xl0-3M, less than 10-3M, less than 5xlO-4M, less than 10-4M, less than 5xl0-5M, less than 10-5M, less than 5xlO-6M, less than 10-6M, less than 5X 10-7M, less than 10-7M, less than 5xl0-8M, less than 10-8M, less than 5xl0-9M, less than 10-9M, or less than 10-10M). Methods for determining the binding affinity of a binding molecule, such as a polypeptide conjugate, to a target molecule or an effector molecule are well known in the art and include, for example, equilibrium dialysis, ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR; e.g., Biacore assays), fluorescent-activated cell sorting (FACS) binding assays and the like.

[0086] According to certain examples, the polypeptide conjugate described herein binds to a P domain of a VP1 protein of a norovirus with high affinity or relatively high affinity. The terms “high affinity” and “relatively high affinity” are used interchangeably herein and refer to a binding affinity between a binding agent and the target molecule of interest with a KD of at least about 10’6M, more particularly at least about 10'7M, even more particularly at least about 10'7M and still even more particularly between about 10'8M to about IO'10M. Again, the determination of such affinity may be conducted under standard competitive binding immunoassay procedures and at standard experimental or environmental conditions (e.g., 25°C and 1 atm), such as those provided herein. In particular examples, the affinity of the polypeptide conjugate described herein has been determined by SPR. In other examples, the affinity of the polypeptide conjugate described herein has been determined by ELISA. In various examples, the affinity of the polypeptide conjugate described herein has been determined by ITC.

[0087] In particular examples, the polypeptide conjugate provided herein has a KD for the P domain (such as a Pl or P2 domain and inclusive of those epitopes described herein for NB26 and NB85) of a VP1 protein of a norovirus (e.g., SEQ ID NOs: 35 to 37) of lower than about 600 nM (e.g., lower than about 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 nM or any range therein), lower than about 300 nM, lower than about 150 nM, lower than about 100 nM, lower than about 50 nM, lower than about 25 nM, lower than about 10 nM, lower than about 5 nM or lower than about 1 nM. More particularly, the polypeptide conjugate suitably has a KD for the P domain of the norovirus of lower than about 50nM, lower than about 25nM, lower than about lOnM, lower than about 5 nM or lower than about 1 nM. According to some examples, the polypeptide conjugate disclosed herein has a KD for the P domain of a VP1 protein of a norovirus virus (e.g., SEQ ID NOs: 35 to 37), such as a VP1 protein of a GII.4 or GIL 10 genotype, of between about 0.1 nM to about 50 nM, more particularly between about 0.2 nM to about 25 nM, even more particularly between about 0.3 nM to about 20 nM, yet even more particularly between about 0.4 nM to about 15 nM or still even more particularly between about 0.5 nM to about 10 nM.

[0088] As described herein, the polypeptide conjugates of the present disclosure surprisingly demonstrate significantly higher affinity for a target norovirus protein than that of their component single domain antibody or nanobody. Accordingly, the polypeptide conjugate may have a KD for the P domain (such as a Pl or P2 domain and inclusive of those epitopes described herein for NB26 and NB85) of a VP1 protein of a norovirus virus (e.g., SEQ ID NOs: 35 to 37) that is at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 fold (or any range therein) less than that of the single domain antibody of the polypeptide conjugate in isolation (e.g., in an unconjugated, native or wildtype form) and in its monovalent form. Put another way, the polypeptide conjugate suitably has an affinity for the P domain of a VP1 protein of a norovirus virus (e.g., SEQ ID NOs: 35 to 37) that is at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 fold (or any range therein) greater than that of the single domain antibody of the polypeptide conjugate in isolation and in its monovalent form. In particular examples, the polypeptide conjugate has a KD for the P domain of a VP1 protein of a norovirus virus (e.g., SEQ ID NOs: 35 to 37) that is between about 5 fold and about 700 fold, more particularly between about 10 fold and about 600 fold, even more particularly between about 15 fold and about 500 fold, still even more particularly between about 20 fold and about 400 fold, or yet even more particularly between about 25 fold and about 250 fold, less than that of the single domain antibody of the polypeptide conjugate in isolation and in its monovalent form.

[0089] According to certain examples, the polypeptide conjugate provided herein does not significantly or substantially bind to a molecule other than the target molecule (e.g., a VP1 protein of a norovirus). The phrase “does not significantly bind to” or “does not substantially bind to” can mean, for example, that the polypeptide conjugate provided herein binds to a molecule other than the target molecule (or to any molecule other than the target molecule) with a binding affinity (e.g., KD) that is at most 50% (e.g., 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less or any range therein) of the binding affinity of said polypeptide conjugate for the target molecule, such as a VP1 protein expressed by a norovirus, under the same physiological or experimental conditions.

[0090] The current disclosure describes a polypeptide conjugate that selectively binds to a norovirus P domain epitope. According to various examples, the polypeptide conjugate provided herein inhibits, ameliorates, treats or prevents a norovirus virus infection by at least partly inhibiting, disrupting or preventing binding or interaction of norovirus viral particles with host- derived co-factors, such as HBGA and bile acids, which are necessary for virus-host cell membrane fusion.

[0091] The polypeptide conjugates of the present disclosure are suitably also capable of at least partly neutralizing a norovirus, such as a norovirus viral particle. As such, in certain examples, the polypeptide conjugate provided herein inhibits, ameliorates, treats or prevents a norovirus virus infection by at least partly neutralizing norovirus viral particles in a subject.

[0092] As such, the polypeptide conjugates of the present disclosure are capable of neutralizing a norovirus (e.g., a norovirus virus particle or a norovirus VLP) and / or blocking HBGA and / or bile acid binding thereto in a virus neutralization assay. In particular examples, the polypeptide conjugate is capable of neutralizing a norovirus or blocking HBGA binding thereto in a neutralization assay at an ICso of less than about 50 nM (e.g., less than about 50, 40, 30, 25, 20, 15, 10, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1, 0.5, 0.2, 0.1, 0.05 nM or any range therein), less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 0.5 nM or less than about 0.1 nM. In other examples, the polypeptide conjugate is capable of neutralizing a norovirus, such as a virus particle or a virus-like particle thereof, or blocking HBGA binding thereto in a neutralization assay at an ICso of between about 0.05 nM and about 25 nM, between about 0.1 nM and about 15 nM, between about 0.1 nM and about 10 nM, between about 0.2 nM and about 10 nM, between about 0.1 nM and about 5 nM, between about 0.2 nM and about 5 nM or between about 0.5 nM and about 5 nM. Suitable virus neutralisation assays include, for example, HBGA blocking assays, a norovirus HIE culture system, such as that described herein, plaque reduction assays, pseudovirus neutralisation assays and microneutralisation assays.

[0093] As described herein, the polypeptide conjugates of the present disclosure surprisingly demonstrate a significantly higher ability to neutralize norovirus, such as in a virus neutralization assay, than that of their component single domain antibody or nanobody. Accordingly, the polypeptide conjugate may be capable of neutralizing a norovirus or blocking HBGA binding thereto in a neutralization assay at an ICso that is at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 fold (or any range therein) less than that of the single domain antibody of the polypeptide conjugate in isolation (e.g., in an unconjugated, native or wildtype form) and in its monovalent form. Put another way, the polypeptide conjugate suitably has a neutralization capacity or capability for a norovirus that is at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 fold (or any range therein) greater than that of the single domain antibody of the polypeptide conjugate in isolation and in its monovalent form. In particular examples, the polypeptide conjugate is capable of neutralizing a norovirus or blocking HBGA binding thereto in a neutralization assay at an ICso that is that is between about 2 fold and about 200 fold, more particularly between about 3 fold and about 100 fold, even more particularly between about 4 fold and about 50 fold, still even more particularly between about 5 fold and about 25 fold, or yet even more particularly between about 5 fold and about 10 fold, less than that of the single domain antibody of the polypeptide conjugate in isolation and in its monovalent form.

[0094] Suitably, the single domain antibody of the polypeptide conjugate described herein includes the CDR1 that comprises the amino acid sequence of RIIFFMYD (SEQ ID NO: 1) or a variant thereof, the CDR2 that comprises the amino acid sequence of QINSDVST (SEQ ID NO: 2) or a variant thereof and the CDR3 that comprises the amino acid sequence of YCNVRRASA (SEQ ID NO: 3) or a variant thereof. In these examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4.

[0095] Suitably, the single domain antibody of the polypeptide conjugate described herein includes the CDR1 that comprises the amino acid sequence of GSIFSIY[A / L] (SEQ ID NO: 5 or 6) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KRED[Y / F]SAYAPPSGS (SEQ ID NO: 8 or 9) or a variant thereof. Referring to various examples, the single domain antibody of the polypeptide conjugate described herein includes the CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8) or a variant thereof. In related examples, the single domain antibody of the polypeptide conjugate described herein includes the CDR1 that comprises the amino acid sequence of GSIFSIYL (SEQ ID NO: 6) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8) or a variant thereof. In further related examples, the single domain antibody of the polypeptide conjugate described herein includes the CDR1 that comprises the amino acid sequence of GSIFSIYL (SEQ ID NO: 6) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof. In other related examples, the single domain antibody of the polypeptide conjugate described herein includes the CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof.

[0096] In some examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 10. In other examples, the single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11.

[0097] According to some examples, the polypeptide conjugate comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 15, or a fragment, variant or derivative thereof. For such examples, the polypeptide conjugate may comprise, consist of or consist essentially of an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12. In other examples, the polypeptide conjugate comprises, consists of or consists essentially of an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. For certain examples, the polypeptide conjugate comprises, consists of or consists essentially of an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14. According to various examples, the polypeptide conjugate comprises, consists of or consists essentially of an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15.

[0098] The polypeptide conjugates, nucleic acids, genetic constructs and host cells described herein may be considered to be isolated. For the purposes of the present disclosure, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

[0099] By “protein” is meant an amino acid polymer. The amino acids may be natural or nonnatural amino acids, D- or L-amino acids as are well understood in the art.

[0100] The term “protein” includes and encompasses “peptide”, which is typically used to describe a protein having no more than fifty (50) amino acids (e.g., no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids and any range therein) and “polypeptide”, which is typically used to describe a protein having more than fifty (50) amino acids.

[0101] As used herein, a protein, polypeptide or peptide “variant” shares a definable amino acid sequence relationship with a reference amino acid sequence. In particular examples, the reference amino acid sequence is that of a CDR1, CDR2 or CDR3 sequence. As such, the reference amino acid sequence may be the amino acid sequence of any one of SEQ ID NOs: 1 to 3 and 5 to 9. According to other examples, the reference amino acid sequence is that of a VHH chain sequence, such as that of any one of SEQ ID NOs: 4, 10 and 11. For some examples, the reference amino acid sequence is that of a FR1, FR2, FR3 or FR4 sequence. In other examples, the reference amino acid sequence is that of a polypeptide conjugate, such as that of any one of SEQ ID NOs: 12 to 15. Referring to alternative examples, the reference amino acid sequence is that of a hinge region, such as that of SEQ ID NO: 16. For various examples, the reference amino acid sequence is that of a Fc region, such as that of any one of SEQ ID NOs: 26 to 34.

[0102] The “variant” protein, polypeptide or peptide may have one or a plurality of amino acids of the reference amino acid sequence deleted, inserted / added or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted, inserted / added or deleted without changing the activity of a single domain antibody, a hinge region and / or a Fc region of the polypeptide conjugate (i.e., conservative substitutions). Accordingly, one or more (e.g., 1, 2, 3, 4, 5 etc) of the residues of a single domain antibody, such as of a CDR (e.g., those defined by SEQ ID NOs: 1 to 3 and 5 to 9) and / or a FR, a hinge region and / or a Fc region of the polypeptide conjugate may be conservatively modified (e.g., by amino acid substitution or deletion) without altering the biological activity, function, or other desired property thereof, such as its affinity or its specificity for an antigen like a P domain of a VP1 protein.

[0103] In some examples, a variant of the polypeptide conjugate provided herein substantially retains the antigen binding ability (i.e., VP1 protein, and more particularly, P2 subdomain thereof, binding ability) of the unmodified or reference single domain antibody thereof. Thus, one or more amino acid residues within the CDR and / or FR regions of a single domain antibody of the polypeptide conjugate of the present disclosure can be deleted or replaced with other amino acid residues, such as those from the same side chain family, and the variant polypeptide conjugate can be tested for retained function (e.g., the ability to specifically bind a P domain of a VP1 protein of a norovirus at high affinity) using the functional assays described herein.

[0104] According to some examples, modifications can be made to decrease the immunogenicity of the single domain antibody of the polypeptide conjugate. For example, one approach is to modify one or more FR residues to that respective FR residue of the corresponding human germline sequence. Another type of framework modification involves modifying one or more residues within the FR and / or CDR regions to remove T cell epitopes to thereby reduce the potential immunogenicity of the single domain antibody of the polypeptide conjugate.

[0105] Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Vai, Feu, and He; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in, for example, Bowie et al., Science 247:1306-1310 (1990).

[0106] Suitably, protein, polypeptide or peptide variants provided herein share at least 70% or 75%, more particularly at least 80% or 85% or even more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% sequence identity with a reference amino acid sequence, such as those set forth in SEQ ID NOs: 1 to 16 and 21 to 53. To this end, variants of the polypeptide conjugates described herein and their respective single domain antibodies, CDRs, hinge regions and Fc regions are contemplated for the present disclosure.

[0107] Accordingly, modifications to the CDR sequences disclosed herein are envisaged. In particular examples, said CDR1 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 1 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In other examples, said CDR1 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 5 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In some examples, said CDR1 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 6 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto.

[0108] In certain examples, said CDR2 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 2 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In particular examples, said CDR2 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 7 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto.

[0109] According to further examples, said CDR3 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 3 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. For some examples, said CDR3 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 8 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto. In other examples, said CDR3 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 9 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identical thereto.

[0110] Larger polypeptide conjugates comprising a plurality of single domain antibodies (e.g., multivalent or multispecific antigen binding molecules) or conjugates thereof are also contemplated by the present disclosure and are described in more detail hereinafter. Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids / proteins may each comprise (1) only one or more portions of a complete nucleic acid / protein sequence that are shared by the nucleic acids / proteins, and (2) one or more portions which are divergent between the nucleic acids / proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of, for example, 6, 9, 12 or 20 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1991, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0111] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) or CLUSTAL Omega

[0112] (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo).

[0113] The present disclosure also provides polypeptide conjugates that contain fragments of the single domain antibodies described herein. As used herein, a “fragment” is a segment, domain, portion or region of a protein or peptide (such as those set forth in SEQ ID NOs: 1 to 16 and 21 to 53) which constitutes less than 100% of the amino acid sequence of the protein or peptide.

[0114] In general, fragments may comprise, consist essentially of or consist of up to 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,

[0115] 137, 138, 139, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,

[0116] 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,

[0117] 315, 320, 325, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,

[0118] 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365 or 366 (inclusive of any range therein) contiguous amino acids of a single domain antibody (such as one of SEQ ID NOs: 4, 10 or 11) or a polypeptide conjugate (such as one of SEQ ID NOs: 12 to 15). In particular examples, the polypeptide conjugate comprises a protein fragment that is or comprises a conserved region or one or more conserved amino acids, such as the CDR1-CDR3, of a single domain antibody. In this regard, one or more residues (e.g., at least 1, 2, 3, 4, 5 etc) of FR1 and / or FR4 at an N- and / or C-terminus of a single domain antibody may not be present in the polypeptide conjugate. In various examples, the polypeptide conjugate comprises a protein fragment which comprises, or is contained within, a single domain antibody, such as those set forth in SEQ ID NOs: 4, 10 or 11.

[0119] Suitably, the fragment substantially retains the antigenic binding ability of the single domain antibody from which the fragment is derived. In this regard, fragments of the present disclosure may retain the CDR1, CDR2 and CDR3 sequences of the single domain antibody. Additionally, fragments of the present disclosure suitably retain at least partly a natural structure and / or conformation of the full-length peptide or protein.

[0120] The present disclosure also contemplates derivatives of the polypeptide conjugate described herein. As used herein, “derivatives” are molecules such as proteins, fragments or variants thereof that have been altered, for example, by conjugation or complexing with other chemical moieties or functional moieties, by post-translational modification (e.g., phosphorylation, acetylation and the like), modification of glycosylation (e.g., adding, removing or altering glycosylation), lipidation and / or inclusion of additional amino acid sequences as would be understood in the art.

[0121] Additional amino acid sequences may include fusion partner amino acid sequences which create a fusion protein. By way of example, fusion partner amino acid sequences may assist in detection and / or purification of the isolated fusion protein. Non-limiting examples include metalbinding (e.g., polyhistidine) fusion partners, maltose binding protein (MBP), Protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g., GFP), polylysine, epitope tags, such as myc, FLAG and haemagglutinin tags. In one particular example, an additional amino acid sequence may comprise one or a plurality of histidine residues at an N and / or C-terminus thereof (e.g., hexa-histidine). The plurality of histidine residues (e.g., polyhistidine) may be a linear sequence of histidine residues or may be branched chain sequences of histidine residues. These additional histidine residues may facilitate purification of the polypeptide conjugate.

[0122] Other derivatives contemplated by the disclosure include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and / or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptide conjugate and variants of the disclosure. In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

[0123] Polypeptide conjugates of the disclosure may be modified via conjugation or complexing with other chemical moieties, by post-translational modification (e.g., phosphorylation, ubiquitination, glycosylation), chemical modification (e.g., cross-linking, acetylation, biotinylation, oxidation or reduction) and / or conjugation with labels (e.g., fluorophores, enzymes, radioactive isotopes) and / or other functional elements (e.g., a half-life extender, a therapeutic agent), as described in more detail below.

[0124] Derivatives of the polypeptide conjugates of the present disclosure suitably retain their ability to bind a P domain of a VP1 protein of a norovirus. In an example, a polypeptide conjugate disclosed herein is conjugated to a label, such as biotin, so as to facilitate coupling to a substrate. Additional C- or N-terminal residues may be used as linkers to conjugate the polypeptide conjugates of the present disclosure to another moiety, or tags that aid the detection of the molecule. Such linkers and tags are well known in the art and include, for example, linker His tags or myc tags.

[0125] Exemplary functional moieties that may be conjugated or otherwise linked to the polypeptide conjugate of the present disclosure include a detectable marker or label, a therapeutic agent, a half-life extender and a nanocarrier, inclusive of combinations thereof.

[0126] In certain examples, the functional moiety is or comprises a half-life extender that may serve to prolong the half-life of the polypeptide conjugate in vivo following administration to a subject. Such half-life extenders may comprise, for example, an antibody, or part thereof, or a protein, or part thereof, that binds or is derived from a serum albumin (e.g., a serum albumin protein, an albumin binding domain). Half-life extenders that may be utilised for the present disclosure include polymers, such as polyethylene glycol (PEG) and starch. Half-life may be increased by at least 1.5 times, more particularly at least 2 times, even more particularly at least 5 times, yet even more particularly at least 10 times or still even more particularly at least 20 times, greater than the half-life of the corresponding polypeptide conjugate of the present disclosure that do not include such a half-life extender. For example, the half-life may be increased by more than 1 hour, more particularly more than 2 hours, even more particularly more than 6 hours, yet even more particularly more than 12 hours, or still even more particularly more than 24, 48 or 72 hours, compared to the polypeptide conjugates of the present disclosure that do not include such a halflife extender.

[0127] According to other examples, the polypeptide conjugates of the present disclosure are labelled with a detectable or functional marker or label. A label can be any molecule that produces or can be induced to produce a signal, including, but not limited to, fluorophores, fluorescent labels, radiolabels, enzymes, chemiluminescent labels, a nuclear magnetic resonance active label or photosensitizers, as described in more detail herein. Thus, binding of the labelled polypeptide conjugate to a P domain of a norovirus may be detected and / or measured by detecting fluorescence, luminescence, radioactivity, enzyme activity or light absorbance thereof.

[0128] For some examples, the polypeptide conjugates of the present disclosure are coupled to a therapeutic agent or moiety (e.g., a small molecule, a protein, a nucleic acid, such as an siRNA), such as a drug, an enzyme, a cytokine (e.g., IL-2, IL- 12, and TNF), a radionuclide or a toxin (e.g., Enterobacter cloacae P-Lactamase, Pseudomonas Exotoxin A, TRAIL and granzyme B). In particular examples, the therapeutic agent is an antiviral agent, as are known in the art. Exemplary antiviral agents include dasabuvir, nitazoxanide, remdesivir and nelfinavir.

[0129] In some examples, the polypeptide conjugates of the present disclosure are linked with, coupled to or otherwise associated with a nanocarrier, such as a liposome, a micelle, a lipid nanoparticle, albumin-based nanoparticles and polymer-based nanoparticles. Because of the binding specificity of single domain antibodies, they may be broadly used in such drug delivery platforms to deliver their cargo (e.g., a therapeutic payload) to its specific location (e.g., a viral particle).

[0130] It is envisaged that all combinations of the polypeptide conjugate, in particular those combinations specifically listed herein, can be used in any therapeutic, diagnostic, or prognostic method or use, such as those hereinafter described.

[0131] The polypeptide conjugates of the present disclosure, inclusive of variants, fragments and / or derivatives thereof, may be produced by any means known in the art, including but not limited to, chemical synthesis, recombinant DNA technology and proteolytic cleavage to produce peptide fragments.

[0132] Chemical synthesis is inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publication WO 99 / 02550 and International Publication WO 97 / 45444.

[0133] Recombinant proteins may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008), in particular Chapters 1, 5 and 6. Typically, recombinant protein preparation includes expression of a nucleic acid encoding the protein in a suitable host cell, as described in more detail herein.

[0134] The present disclosure further contemplates humanized or at least partly humanized (also referred to as “humaneered”) versions of the single domain antibodies provided herein. Accordingly, in some examples, the single domain antibody of the polypeptide conjugate provided herein is a humanized or substantially humanized single domain antibody.

[0135] By “humanized” is meant the amino acid sequence of the single domain antibody is mutated or modified so that immunogenicity upon administration in human patients is reduced, minor or non-existent (e.g., a single domain antibody that originated from a species other than human that has had immunogenic or potentially immunogenic amino acid residues replaced with amino acids that are less immunogenic or not immunogenic in the context of a single domain antibody administered to a human subject). Accordingly, humanized single domain antibodies should be substantially non-immunogenic in humans, but retain the affinity and activity of the wild-type, unmodified or camelid single domain antibody (e.g., the single domain antibodies of SEQ ID NOs: 4, 10 or 11).

[0136] Any method known in the art for creating humanized antibodies are envisaged herein, including but not limited to the humanizing technology of KaloBios Pharmaceuticals and that described in Vincke et al. (JBC 2008). By way of example, humanising a single domain antibody generally comprises a step of replacing one or more of the camelid- or alpaca-derived amino acid residues with their human counterpart as found in a corresponding human consensus sequence, without that single domain antibody losing its typical character or biological function. Suitably, humanization does not significantly affect the antigen binding capacity of the resulting humanized single domain antibody. Notwithstanding this, humanizing modifications or mutations may be made in one or more CDRs and / or FRs of the single domain antibodies. In particular examples, the humanized single domain antibody may contain one or more fully human FR sequences, as are known in the art.

[0137] Fragment crystallizable regions

[0138] By “Fc”, “Fc domain” or “Fc region”, as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, an Fc region refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and optionally at least part of the flexible hinge N-terminal to these domains. These terms as used herein also mean a polypeptide that comprises all or part of an Fc region. Suitably, the Fc region is fused or conjugated to the N-terminus or the C-terminus of the single domain antibody of the polypeptide conjugate. In certain examples, the Fc region is fused to the C-terminus of the single domain antibody of the polypeptide conjugate.

[0139] It is contemplated that the Fc region provided herein can be a naturally or non-naturally occurring Fc sequence or a fragment or portion thereof. Irrespective of whether the Fc region is native or modified, it suitably at least partly retains one or more Fc region-associated or mediated functions, such as antibody-dependent cellular cytotoxicity (ADCC), complement binding, activation and complement-dependent cytotoxicity (CDC), phagocytosis / clearance of antigenantibody complexes, cytokine release, and half-life / clearance rate of the polypeptide conjugate. In some examples, the variant Fc region has altered effector activity relative to a corresponding, wildtype or native Fc region.

[0140] Moreover, the Fc region may be of any native or modified form or sequence as are known in the art. The Fc region can be derived from the CH2 region, CH3 region, CH4 region, and / or hinge region(s) of any one or more immunoglobulin classes. To this end, the Fc region may be derived from any immunoglobulin class (e.g., IgA, IgD, IgG, IgE or IgM), subclass (e.g., IgAl, IgA2, IgGl, IgG2, IgG3 or IgG4) or species (e.g., human, mouse, canine, bovine etc). In some examples, the Fc region is derived from an IgA immunoglobulin, including subclasses IgAl and / or IgA2. In certain examples, the Fc region is derived from an IgD immunoglobulin. In particular examples, the Fc region is derived from an IgE immunoglobulin. In some examples, the Fc region is derived from an IgG immunoglobulin, including subclasses IgGl, IgG2, IgG2, IgG3, and / or IgG4. In certain examples, the Fc region is derived from an IgM immunoglobulin. For some examples, the Fc region is derived from an IgG class, and more particularly an IgGl subclass, as are known in the art.

[0141] It is contemplated that the single domain antibodies described herein may be conjugated with any Fc region known in the art to form a polypeptide conjugate. Nonlimiting examples of amino acid sequences of Fc regions are provided in SEQ ID NOs: 26 to 34. Referring to some examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 26, or a fragment, variant or derivative thereof. For other examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 27, or a fragment, variant or derivative thereof. In certain examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO:

[0142] 28, or a fragment, variant or derivative thereof. Referring to particular examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO:

[0143] 29, or a fragment, variant or derivative thereof. According to various examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO:

[0144] 30, or a fragment, variant or derivative thereof. Referring to other examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO:

[0145] 31, or a fragment, variant or derivative thereof. In some examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 32, or a fragment, variant or derivative thereof. For other examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 33, or a fragment, variant or derivative thereof. In further examples, the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 34, or a fragment, variant or derivative thereof.

[0146] Linkers

[0147] Suitably, the single domain antibody of the polypeptide conjugate is conjugated, joined, connected or otherwise linked to the Fc region or Fc domain by way of a linker. The linker may comprise any suitable amino acid sequence known in the art. In alternative examples, the single domain antibody of the polypeptide conjugate is directly conjugated or linked to the Fc region or Fc domain (i.e., no linker is present).

[0148] In various examples, the single domain antibody is conjugated, connected or otherwise linked to the Fc region by a hinge region or a portion or fragment thereof. The term “hinge region” denotes the part of an antibody heavy chain polypeptide that joins in a wild-type antibody heavy chain the CHI domain and the CH2 domain (e.g., from about position 216 to about position 230 according to the EU number system of Kabat, or from about position 226 to about position 230 according to the EU number system of Kabat). The hinge regions of other IgG subclasses can be determined by aligning with the hinge-region cysteine residues of the IgGl subclass sequence. The hinge region is normally a dimeric molecule consisting of two polypeptides with identical amino acid sequence. The hinge region generally comprises about 5 to 25 amino acid residues and is flexible allowing the antigen binding regions to move independently. The hinge region can be subdivided into three domains: the upper, the middle, and the lower hinge domain (see e.g. Roux, et al., J. Immunol. 161 (1998) 4083).

[0149] The hinge region provided herein can be or comprise a naturally or non-naturally occurring hinge sequence or a fragment or portion thereof. A native hinge region is the hinge region that would normally be found between Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge region that differs in length and / or amino acid composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may comprise a complete hinge region derived from an antibody of a different class or subclass from that of the heavy chain Fc region. Alternatively, the modified hinge region may comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region. In a further alternative, the natural hinge region may be altered by converting one or more cysteine or other residues into neutral residues, such as serine or alanine, or by converting suitably placed residues into cysteine residues. By such means, the number of cysteine residues in the hinge region may be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to possess desired properties such as length, cysteine composition and flexibility.

[0150] A number of hinge regions, including modified versions thereof, have been described, for example, in US5,677,425, WO 1999 / 15549, W02005 / 003170, W02005 / 003169,

[0151] W02005 / 003170, WO1998 / 25971 and W02005 / 003171, which are incorporated herein by reference.

[0152] Suitably, the hinge region comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 16, or a fragment, variant or derivative thereof.

[0153] Encoding nucleic acids

[0154] The present disclosure also provides an isolated nucleic acid encoding the polypeptide conjugate described herein.

[0155] The term “nucleic acid” as used herein designates single- or double- stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. Nucleic acids may also be DNA-RNA hybrids. A nucleic acid comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as modified purines (for example inosine, methylinosine and methyladenosine) and modified pyrimidines (for example thiouridine and methylcytosine). As used herein, a “ polynucleotide ” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “oligonucleotide ” has less than eighty (80) contiguous nucleotides. A “primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid “template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™ A “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

[0156] Suitably, the isolated nucleic acid encodes a polypeptide conjugate, such as a polypeptide conjugate comprising:

[0157] (a) a single domain antibody that is directed against a P domain of a norovirus, wherein the single domain antibody comprises:

[0158] (i) a CDR1 that comprises the amino acid sequence of RIIFFMYD (SEQ ID NO: 1) or a variant thereof, a CDR2 that comprises the amino acid sequence of QINSDVST (SEQ ID NO: 2) or a variant thereof and a CDR3 that comprises the amino acid sequence of YCNVRRASA (SEQ ID NO: 3) or a variant thereof; or

[0159] (ii) a CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5), GSIFSIYL (SEQ ID NO: 6) or a variant thereof, a CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and a CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8), KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof; and

[0160] (b) an Fc region of an immunoglobulin, or a fragment, variant or derivative thereof.

[0161] For such examples, the single domain antibody of the polypeptide conjugate suitably includes the CDR1 that comprises the amino acid sequence of GSIFSIY[A / L] (SEQ ID NO: 5 or 6) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KRED[Y / F]SAYAPPSGS (SEQ ID NO: 8 or 9) or a variant thereof. Referring to other examples, the single domain antibody of the polypeptide conjugate includes the CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8) or a variant thereof. In related examples, the single domain antibody of the polypeptide conjugate includes the CDR1 that comprises the amino acid sequence of GSIFSIYL (SEQ ID NO: 6) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8) or a variant thereof. In further related examples, the single domain antibody of the polypeptide conjugate includes the CDR1 that comprises the amino acid sequence of GSIFSIYL (SEQ ID NO: 6) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof. In other related examples, the single domain antibody of the polypeptide conjugate includes the CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof.

[0162] In some examples, the isolated nucleic acid encodes a polypeptide conjugate comprising, consisting essentially of or consisting of the amino acid sequence set forth in any of SEQ ID NOs: 12 to 15, or a fragment, derivative or variant thereof.

[0163] Suitably, the isolated nucleic acid encoding the polypeptide conjugate comprises, consists essentially of or consists of the nucleotide sequence set forth in any of SEQ ID NOs: 17 to 20, or a fragment, derivative or variant thereof. In some examples, the isolated nucleic acid encoding the polypeptide conjugate comprises, consists essentially of or consists of the nucleotide sequence set forth in SEQ ID NO: 17, or a fragment, derivative or variant thereof. Referring to other examples, the isolated nucleic acid encoding the polypeptide conjugate comprises, consists essentially of or consists of the nucleotide sequence set forth in SEQ ID NO: 18, or a fragment, derivative or variant thereof. For certain examples, the isolated nucleic acid encoding the polypeptide conjugate comprises, consists essentially of or consists of the nucleotide sequence set forth in SEQ ID NO: 19, or a fragment, derivative or variant thereof. According to particular examples, the isolated nucleic acid encoding the polypeptide conjugate comprises, consists essentially of or consists of the nucleotide sequence set forth in SEQ ID NO: 20, or a fragment, derivative or variant thereof.

[0164] Also contemplated are fragments and variants of the isolated nucleic acid. Variants may comprise a nucleotide sequence at least 70%, at least 75%, preferably at least 80%, at least 85%, more preferably at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with any nucleotide sequence encoding the polypeptide conjugate of the present disclosure (e.g., SEQ ID NOs: 17 to 20).

[0165] Fragments of the isolated nucleic acid may comprise or consist of up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95-99% of the contiguous nucleotides present in any nucleotide sequence encoding the polypeptide conjugate of the present disclosure, such that they encode at least a portion of the polypeptide conjugate. In general, fragments may comprise, consist essentially of or consist of up to 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315, 330, 345, 360, 375, 390, 405, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1020, 1040, 1060 or 1080 (or any range therein) contiguous nucleic acids that encode a portion of a polypeptide conjugate described herein (e.g., SEQ ID NOs: 12 to 15 and 54 to 57).

[0166] The present disclosure also provides nucleic acids that have been modified such as by taking advantage of codon sequence redundancy. In a more particular example, codon usage may be modified to optimize expression of a nucleic acid in a particular organism or cell type.

[0167] The isolated nucleic acids disclosed herein can be conveniently prepared using standard protocols such as those described in Chapter 2 and Chapter 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008). Nucleic acids of the present disclosure may be isolated, detected and / or subjected to recombinant DNA technology using nucleic acid sequence amplification techniques or synthetic production techniques, as are known in the art. Suitable nucleic acid amplification techniques covering both thermal and isothermal methods are well known to the skilled addressee, and include polymerase chain reaction (PCR); strand displacement amplification (SDA); rolling circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-P replicase amplification, recombinase polymerase amplification (RPA) and helicase-dependent amplification, although without limitation thereto.

[0168] Genetic constructs

[0169] The present disclosure also provides a genetic construct comprising the isolated nucleic acid hereinbefore described. The genetic construct may be a vector.

[0170] In particular examples, the genetic construct comprises the isolated nucleic acid operably linked or connected to one or more other genetic components, including heterologous genetic components. A genetic construct may be suitable for therapeutic delivery of the isolated nucleic acid or for recombinant production of the polypeptide conjugate of the disclosure in a host cell.

[0171] Broadly, the genetic construct can be in the form of, or comprises genetic components of, a plasmid, bacteriophage, a cosmid, a yeast or bacterial artificial chromosome as are well understood in the art. Genetic constructs may be suitable for maintenance and propagation of the isolated nucleic acid in bacteria or other host cells, for manipulation by recombinant DNA technology and / or expression of the nucleic acid or an encoded protein of the present disclosure.

[0172] For the purposes of host cell expression, the genetic construct is an expression construct. Suitably, the expression construct comprises the nucleic acid of the present disclosure operably linked to one or more additional sequences, such as one or more heterologous sequences, in an expression vector. An “expression vector" may be either a self-replicating extra-chromosomal vector, such as a plasmid, or a vector that integrates into a host genome.

[0173] By “operably linked” is meant that said additional nucleotide sequence(s) is / are positioned relative to the nucleic acid of the present disclosure preferably to initiate, regulate or otherwise control transcription.

[0174] Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

[0175] Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, polyadenylation sequences, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive, repressible or inducible promoters as known in the art are contemplated by the present disclosure.

[0176] The expression construct may also include an additional nucleotide sequence encoding a fusion partner (typically provided by the expression vector) so that the recombinant protein is expressed as a fusion protein.

[0177] The expression construct may also include an additional nucleotide sequence encoding a selection marker such as ampR, neoRor kanR, although without limitation thereto.

[0178] Host cells

[0179] The present disclosure also provides a host cell transformed with a nucleic acid molecule or a genetic construct described herein. The host cell may be an isolated host cell or a cell in vitro.

[0180] Suitable host cells for expression may be prokaryotic or eukaryotic. For example, suitable host cells may include but are not limited to mammalian cells (e.g. HeLa. Cos, NIH-3T3, HEK293T, Jurkat, CHO cells), yeast cells (e.g. Saccharomyces cerevisiae), insect cells (e.g., Sf9, Trichoplusia ni) utilized with or without a baculovirus expression system, plant cells (e.g. Chlamydomonas reinhardtii, Phaeodactylum tricornutum) or bacterial cells, such as E. coli. Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, as for example described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-2015), in particular Chapters 9 and 16.

[0181] Methods of production

[0182] Related aspects of the present disclosure provide a method of producing the polypeptide conjugate described herein, including the steps of; (i) culturing the host cell disclosed herein; and (ii) isolating the polypeptide conjugate from said host cell cultured in step (i). In this regard, the recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols, such as those hereinbefore provided.

[0183] The present disclosure further provides a polypeptide conjugate produced by the method described herein.

[0184] Pharmaceutical compositions

[0185] Further aspects of the present disclosure provide a composition comprising the polypeptide conjugate described herein, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0186] By “pharmaceutically-acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water,

[0187] A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ. USA, 1991), which is incorporated herein by reference.

[0188] In some examples, the present composition is in the form of a diagnostic composition.

[0189] In other examples, the present composition is in the form of a therapeutic composition.

[0190] A therapeutically effective amount of a composition comprising a polypeptide conjugate may be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the frequency of administration is dependent on the preparation applied, the subject being treated, the severity of the viral infection, and the manner of administration of the therapy or composition.

[0191] Any safe route of administration may be employed for administering the polypeptide conjugate described herein. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids arid certain cellulose derivatives such as hydroxypropylmethyl cellulose, in addition, the controlled release may be achieved by using other polymer matrices, liposomes and / or microspheres. In particular examples, the composition is capable of being or configured or adapted to be administered orally to a subject in need thereof. For such examples, the composition is suitably enterically coated.

[0192] Compositions of the present disclosure suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water- in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the therapeutic agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0193] The above compositions may be administered in a manner compatible with the dosage formulation, and in such an amount as is effective to prophylactically and / or therapeutically treat noroviral infections, and / or diseases, disorders or conditions associated therewith and / or alleviate symptoms associated therewith. The dose administered to a patient, in the context of the present disclosure, should be sufficient to achieve a beneficial response in a patient over time such as a reduction in a level of viral shedding in their bodily fluids (e.g., blood, urine, saliva). The quantity of the therapeutic agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the therapeutic agent(s) required to be administered will depend on the judgement of the clinician. The total dose required for each treatment may be administered by multiple doses or in a single dose. In any event, suitable dosages of the therapeutic agents described herein may be readily determined by those skilled in the art. Such dosages may be in the order of nanograms to milligrams of the therapeutic agents of the disclosure.

[0194] Suitably, the composition further comprises one or more further or additional therapeutic agents, such as an anti-inflammatory agent (e.g., NSAIDs, corticosteroids) and / or an antiviral agent (e.g., a milk oligosaccharide, NB-2, NB-7, NB-45, NB-76, dasabuvir, nitazoxanide, remdesivir and nelfinavir). According to some examples, the composition further comprises a milk oligosaccharide, such as 2’ -FL.

[0195] Suitably, the composition further includes a further single domain antibody, inclusive of a further single domain antibody conjugated to an Fc region (i.e., in the form of a polypeptide conjugate described herein), that is directed against a norovirus, such as one that binds to the P domain (e.g., a Pl domain and / or a P2 domain) of a VP1 protein of a norovirus. A number of antinorovirus antibodies and nanobodies binding to a norovirus VP1 protein have been published, such as MAM4-1418, binding to residues 418 to 426 and 526 to 534 thereof (Shiota T et al, J Virol. 2007 Nov;81(22): 12298-306); NV23, NS22, and F120, binding to residues 453-472 thereof, NS 14, binding to residues 473-494 thereof, 8C7, binding to residues 451-489 thereof (Crawford SE et al, Clin Vaccine Immunol. 2015 Feb;22(2): 168-77; Kou B et al., Clin Vaccine Immunol. 2015 Feb;22(2): 160-7), 5B18, binding to residues 433, 496, 533-535 thereof (Hansman GS et al. J Virol. 2012 Apr;86(7):3635-46), NV3901 and NV3912, binding to residues 454-520 thereof (Parker TD et al, J Virol. 2005 Jun;79(12):7402-9), 1B4 and 1F6, binding to residues 45-55 thereof (Yoda T et al, J Clin Microbiol. 2003 Jun;41(6):2367-71), N2C3, binding to residues 55-60 thereof (Li X et al, Virus Res. 2009 Mar;140(l-2): 188-93, Li X et al, Virus Res. 2010 Aug;151(2): 142- 7), an antibody binding to residues 52-56 thereof (Gabriel I. Parra et al. PLoS One. 2013; 8(6): e67592), NB-2, NB-7, NB-45 and NB-76 (PCT / AU2024 / 050213; Kher, et al., Journal of Virology. Vol. 97, No. 4, pages 1-23), which are incorporated by reference herein.

[0196] In various examples, the composition further comprises a NB-2 single domain antibody or a functional variant, fragment or derivative thereof. Accordingly, in some examples, the further single domain antibody comprises the CDR1 that comprises the amino acid sequence of ASGRFFSSYA (SEQ ID NO: 42) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISWSGGST (SEQ ID NO: 43) or a variant thereof and the CDR3 that comprises the amino acid sequence of AREGAYYPDSYYRTVRYD (SEQ ID NO: 44) or a variant thereof. More particularly, the further single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 38, or a fragment, variant or derivative thereof.

[0197] In some examples, the composition further comprises a NB-7 single domain antibody or a functional variant, fragment or derivative thereof. As such, in other examples, the further single domain antibody comprises the CDR1 that comprises the amino acid sequence of ASGRTFSSY (SEQ ID NO: 45) or a variant thereof, the CDR2 that comprises the amino acid sequence of TGSGD (SEQ ID NO: 46) or a variant thereof and the CDR3 that comprises the amino acid sequence of YRTGGPPQW (SEQ ID NO: 47) or a variant thereof. More particularly, the further single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 39, or a fragment, variant or derivative thereof.

[0198] In other examples, the composition further comprises a NB-45 single domain antibody or a functional variant, fragment or derivative thereof. Accordingly, in such examples, the further single domain antibody comprises the CDR1 that comprises the amino acid sequence of RTDSEST (SEQ ID NO: 48) or a variant thereof, the CDR2 that comprises the amino acid sequence of WRYA (SEQ ID NO: 49) or a variant thereof and the CDR3 that comprises the amino acid sequence of RYIYGSLSDSGSYDN (SEQ ID NO: 50) or a variant thereof. More particularly, the further single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 40, or a fragment, variant or derivative thereof.

[0199] In further examples, the composition further comprises a NB-76 single domain antibody or a functional variant, fragment or derivative thereof. As such, in various examples, the further single domain antibody comprises the CDR1 that comprises the amino acid sequence of SGTIFSIDA (SEQ ID NO: 51) or a variant thereof, the CDR2 that comprises the amino acid sequence of QAPGKQRE (SEQ ID NO: 52) or a variant thereof and the CDR3 that comprises the amino acid sequence of AKPPTYYSLEPWGKGT (SEQ ID NO: 53) or a variant thereof. More particularly, the further single domain antibody suitably comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 41, or a fragment, variant or derivative thereof.

[0200] Noroviruses

[0201] Noroviruses belong to a genetically diverse group of non-enveloped, single- stranded RNA viruses of the Caliciviridae family. Human noroviruses have a single- stranded, positive sense RNA genome of ~7.7 kb. The norovirus genome generally contains three open reading frames (ORFs), where ORF1 encodes the non- structural proteins and includes the protease and RNA dependent RNA polymerase (RdRp), ORF2 encodes the capsid protein (VP1), and ORF3 encodes a small protein (VP2). The VP1 protein can be divided into two domains, a shell (S) domain and a protruding (P) domain, where the S domain surrounds the viral RNA, and the P domain, which can be further subdivided into Pl and P2 subdomains, contains the determinants for co-factor binding and antibody recognition. The P domain is generally in a dimeric form that protrudes from the shell of the norovirus capsid and engages HBGAs (typically two HBGA binding sites per P domain dimer), that are recognized co-factors and believed to be critical for most norovirus infections. Based on the ABH- and Lewis-HBGA types, at least nine different HBGA types have been found to interact with human noroviruses, although HBGA types and binding sites can vary among genogroups and genotypes.

[0202] For the present disclosure, it is envisaged that the term “norovirus” comprises any norovirus, irrespective of strain or origin. Suitably, the term “norovirus” encompasses those norovirus strains of Genogroup I, Genogroup II, Genogroup III, Genogroup IV, and Genogroup V (abbreviated as GI, GII, Gill, GIV or GV, respectively). In certain examples, the norovirus provided herein comprises a norovirus strain selected from the group consisting of:

[0203] (i) Genogroup I genotype 1 (abbreviated as GI.l), GI.2, GI.3, GI.4, GI.5, GI.6, GI.7, GI.8, GI.9, GI.10, GI.l l, GI.12, GI.13, GI.14, GI.15. GI.16 and / or GI.17;

[0204] (ii) Genogroup II genotype 1 (abbreviated as GII.l), GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.8, GII.9, GII.10, GII.l l, GII.12, GII.13, GII.14, GII.15, GII.16, GII.17, GII.18, GII.19, GII.20, GII.21, GII.22, GII.23, GII.24, GII.25, GII.26, GII.27 and / or GII.NA1;

[0205] (iii) Genogroup III genotype 1 (abbreviated as GIII.l), GIII.2, GIII.3, and / or GIII.4;

[0206] (iv) Genogroup IV genotype 1 (abbreviated as GIV.l), GIV.2, GIV.3, and / or GIV.4;

[0207] (v) Genogroup V genotype 1 (abbreviated as GV.l), GV.2, GV.3, and / or GV.4;

[0208] (vi) Genogroup VIII genotype (abbreviated as GVIII); and / or combinations of 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more of any of the above noroviruses from different genogroup s and / or different genotypes.

[0209] Suitably, the norovirus described herein is of a GII genogroup. More particularly, the norovirus suitably comprises a norovirus strain or genotype selected from the group consisting of a GII.l genotype, a GII.4 genotype, a GII.8 genotype, a GII.9 genotype, a GII.10 genotype, a GII.14 genotype, a GII.17 genotype, a GII.23 genotype, a GII.24 genotype, a GII.26 genotype, a GII.27 genotype or a GII.NA1 genotype. Even more particularly, the norovirus is suitably of a GII.4 genotype, a GII.10 genotype or a GII.17 genotype. According to various examples, the norovirus provided herein is of a GII.4 genotype, such as the GII.4 Sydney-2012 strain. In other examples, the norovirus provided herein is of a GII.10 genotype, such as the GII.10 Vietnam 026 strain. For various examples, the norovirus provided herein is of a GII.17 genotype, such as the GII.17 Kawasaki3O8 strain. According to other examples, the norovirus provided herein is of a GII.9 genotype, such as the GII.9-VA97207 strain. For some examples, the norovirus provided herein is of a GII.23 genotype, such as the GII.23-Eoretol847 strain. In certain examples, the norovirus provided herein is of a GII.27 genotype, such as the GII.27-Eoreto0959 strain.

[0210] The terms “VP1”, “VP1 protein”, “VP1 polypeptide” or “capsid protein” as used herein includes any of the recombinant or naturally-occurring forms of a noroviral VP1 or capsid protein, or variants or homologs thereof that at least partly maintain or retain VP1 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to a wildtype or naturally occurring VP1 protein sequence). In some examples, the variants or homologs have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 continuous amino acid portion), such as a P domain or P2 subdomain thereof, compared to a wildtype or naturally occurring VP1 protein sequence (e.g., SEQ ID NOs: 35 to 37). According to some examples, the VP1 protein is substantially identical to the protein identified by the UniProt accession number K4LM89 (norovirus strain GII.4 Sydney- 2012), Q5F4T5 (Norwalk virus; norovirus strain GIL 10 Vietnam 026) or A0A0E4B 1P1 (norovirus strain GIL 17 Kawasaki3O8). In other examples, the VP1 protein is substantially identical to the protein identified by the Protein Database accession number 9EDM (norovirus strain GII.9- VA97207), 9EDN (norovirus strain GII.23 -Loreto 1847) or 9EDO (norovirus strain GII.27- Loreto0959).

[0211] Sequences of a VP1 protein for a range of norovirus strains are publicly available. Exemplary amino acid sequences are set forth in SEQ ID NOs: 35 to 37. Thus, the VP1 protein amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 35 or a fragment or derivative thereof. Alternatively, the VP1 protein amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 36 or a fragment or derivative thereof. In other examples, the VP1 protein amino acid sequence may be a protein which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 37 or a fragment or derivative thereof.

[0212] Suitably, the single domain antibody described herein is directed against a P domain of a norovirus, such as a P domain of a VP1 protein. The expression “directed against a P domain of a norovirus” is intended to mean that the single domain antibody, and by extension the polypeptide conjugate, of the present disclosure is capable of selectively or specifically binding to or interacting with and / or has been raised against an epitope of the P domain of a norovirus. Accordingly, the single domain antibody or polypeptide conjugate may be described as specific or selective for the P domain of noroviruses, that is to say that it binds to the P domain of this virus to the exclusion of any other molecule or protein. In particular examples, the single domain antibody or polypeptide conjugate herein binds or interacts with one monomer of the P domain. In other examples, the single domain antibody or polypeptide conjugate herein binds or interacts with both monomers of the P domain.

[0213] Typically, a P domain of a VP1 protein of a norovirus comprises and extends from residue 222, 223, 224 or 225 thereof to the C-terminal end of said VP1 protein. In particular examples, a P domain comprises, consists of or consists essentially of residues 224 to 540 of a VP1 protein of a norovirus, such as a GII.4 strain of a norovirus. More particularly, a P domain may comprise, consist of or consist essentially of residues 224 to 540 of a VP1 protein having an amino acid sequence set forth in SEQ ID NO: 35. In certain examples, a P domain comprises, consists of or consists essentially of residues 222 to 548 or 549 of a VP1 protein of a norovirus, such as a GIL 10 strain of a norovirus. More particularly, a P domain may comprise, consist of or consist essentially of residues 222 to 548 of a VP1 protein having an amino acid sequence set forth in SEQ ID NO: 36. In other examples, a P domain comprises, consists of or consists essentially of residues 225 to 540 of a VP1 protein of a norovirus, such as a GII.17 strain of a norovirus. More particularly, a P domain may comprise, consist of or consist essentially of residues 225 to 540 of a VP1 protein having an amino acid sequence set forth in SEQ ID NO: 37. Even more particularly, a P domain may comprise, consist of or consist essentially of an amino acid sequence set forth in Figure 7 or Figure 10 of the present disclosure.

[0214] It is contemplated that residue or amino acid numbering herein can be based on any full length sequence of a VP1 protein of a norovirus known in the art (e.g., an amino acid sequence set forth in any one of SEQ ID NOs: 35 to 37 or provided in Figure 7 or Figure 10; i.e., the amino acid numbering is based on the first methionine (methionine in position 1; MET-1) being the first residue). In particular examples, residue numbering is based on a VP1 protein that is derived from a GII.4 strain (e.g., SEQ ID NO: 35). According to other examples, residue numbering is based on a VP1 protein that is derived from a GII.10 strain (e.g., SEQ ID NO: 36). For certain examples, residue numbering is based on a VP1 protein that is derived from a GII.17 strain (e.g., SEQ ID NO: 37).

[0215] Given the sequence similarity between the respective VP1 proteins and P domains of different norovirus genogroups, genotypes and strains, the single domain antibody or polypeptide conjugate of the present disclosure may also be capable of selectively or specifically binding to a P domain of a VP1 protein of a heterologous genogroup, genotype and / or strain of a norovirus (e.g., a genotype, genogroup or strain of norovirus that is different to that genotype, genogroup or strain from which the P domain of the VP1 protein has been utilized to raise or generate the single domain antibody in question). Accordingly, in some examples, the single domain antibodies and polypeptide conjugates of the present disclosure may be able to prevent or treat noroviral infections associated with one or more other genogroups, genotypes, variants and / or strains of norovirus in addition to that genogroup, genotype, variant and / or strain of norovirus from which the single domain antibody in question has been raised or generated.

[0216] Methods for preventing and treating noroviral infections

[0217] In view of the foregoing, the polypeptide conjugates, nucleic acids, genetic constructs, host cells and compositions described herein can be utilised to prevent, ameliorate and / or treat noroviral infections, such as those caused by a GII genogroup, and more particularly a GII.4 genotype, a GII.10 genotype or a GII.17 genotype, and / or diseases, disorders or conditions associated therewith.

[0218] In one broad form, the present disclosure provides a method of inhibiting or preventing binding of a norovirus, such as a viral particle thereof, to a HBGAs and / or a bile acid in a subject, said method including the step of administering to the subject a therapeutically effective amount of the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein to thereby inhibit or prevent binding of the norovirus to the HBGA and / or the bile acid in the subject.

[0219] In a related form, the present disclosure relates to the use of the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein in the manufacture of a medicament for inhibiting or preventing binding of a norovirus to a HBGA and / or a bile acid in a subject.

[0220] In yet a further related form, the present disclosure relates to the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein for use in a method of inhibiting or preventing binding of a norovirus to a HBGA and / or a bile acid in a subject.

[0221] In another broad form, the present disclosure relates to a method of inactivating or neutralising a norovirus, such as a virus particle thereof, in a subject, said method including the step of administering to the subject a therapeutically effective amount of the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein to thereby inactivate or neutralize the norovirus in the subject.

[0222] In a related form, the present disclosure relates to the use of the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein in the manufacture of a medicament for inactivating or neutralising a norovirus, such as a virus particle thereof, in a subject.

[0223] In yet a further related form, the present disclosure relates to the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein for use in a method of inactivating or neutralising a norovirus, such as a virus particle thereof, in a subject.

[0224] In another broad form, the present disclosure relates to the use of a polypeptide conjugate, a composition, an isolated nucleic acid, a genetic construct, a host cell described herein for therapy.

[0225] Accordingly, there is provided herein a method of treating and / or preventing a norovirus infection, and / or a disease, disorder or condition associated therewith in a subject, said method including the step of administering to the subject a therapeutically effective amount of the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein to thereby treat or prevent the norovirus infection and / or disease, disorder or condition associated therewith in the subject.

[0226] In another form, there is provided the use of the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein in the manufacture of a medicament for the treatment and / or prevention of a norovirus infection and / or a disease, disorder or condition associated therewith in a subject.

[0227] In yet a further related form, the present disclosure relates to the polypeptide conjugate, the isolated nucleic acid, the genetic construct, the host cell or the composition disclosed herein for use in the treatment and / or prevention of a norovirus infection and / or a disease, disorder or condition associated therewith in a subject.

[0228] In relation to the above aspects of the present disclosure, the norovirus infection can be the result of any norovirus genogroup, genotype, strain or variant as are known in the art and provided herein. Suitably, the norovirus infection is the result of or caused by of a GII genogroup, such as of a GII.l genotype, a GII.4 genotype, a GII.8 genotype, a GII.9 genotype, a GII.10 genotype, a GII.14 genotype, a GII.17 genotype, a GII.23 genotype, a GII.24 genotype, a GII.26 genotype, a GII.27 genotype or a GII.NA1 genotype. In particular examples, the norovirus infection can be a GII.4 norovirus infection and / or a disease, disorder or condition associated therewith. In further examples, the norovirus infection can be a GII.10 norovirus infection and / or a disease, disorder or condition associated therewith. In other examples, the norovirus infection can be a GII.17 norovirus infection and / or a disease, disorder or condition associated therewith. For some examples, the norovirus infection can be a GII.l norovirus infection and / or a disease, disorder or condition associated therewith. Referring to other examples, the norovirus infection can be a GII.9 norovirus infection and / or a disease, disorder or condition associated therewith. In some examples, the norovirus infection can be a GII.23 norovirus infection and / or a disease, disorder or condition associated therewith. For particular examples, the norovirus infection can be a GII.27 norovirus infection and / or a disease, disorder or condition associated therewith.

[0229] As used herein, “treating” (or “treat” or “treatment”) refers to a therapeutic intervention that ameliorates a sign or symptom of a disease, disorder or condition characterized by a norovirus infection, after it has begun to develop. The term “ameliorating”, with, reference to such diseases, disorders or conditions, refers to any observable beneficial effect of the treatment. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0230] As used herein, “preventing” (or “prevent” or “prevention”) refers to a course of action (such as administering a therapeutically effective amount of the single domain antibody) initiated prior to the onset of a symptom, aspect, or characteristic of a norovirus infection so as to prevent or reduce the symptom, aspect, or characteristic. It is to be understood that such preventing need not be absolute to be beneficial to a subject.

[0231] Prophylactic administration of the polypeptide conjugate, isolated nucleic acids, genetic constructs, host cells and compositions described herein is also envisaged for the present disclosure, particularly in respect of patients who have had prior exposure or contact with an animal or human known or suspected to have been infected with a norovirus. A “prophylactic” treatment, such as passive immunisation, is generally a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition or exhibits only early signs for the purpose of decreasing the risk of developing a symptom, aspect, or characteristic thereof.

[0232] By “administration” is meant the introduction of a composition (e.g., a composition comprising a polypeptide conjugate) or agent (e.g., a polypeptide conjugate, an isolated nucleic acid, a host cell, a genetic construct) into a subject by a chosen route. In some examples, the therapeutically effective amount of the composition or agent is administered subcutaneously. In other examples, the therapeutically effective amount of the composition or agent is administered intramuscularly. In various examples, the therapeutically effective amount of the composition or agent is administered intravenously. In some examples, the therapeutically effective amount of the composition or agent is administered by inhalation. In particular examples, the therapeutically effective amount of the composition or agent is administered by lumbar puncture (spinal tap). In particular examples, the therapeutically effective amount of the composition or agent is administered orally.

[0233] The term “therapeutically effective amount” describes a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this can be the amount of a composition comprising the polypeptide conjugate, composition, genetic construct, host cell or isolated nucleic acid necessary to reduce, alleviate and / or prevent a norovirus infection. In some examples, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of the norovirus infection. In other examples, a “therapeutically effective amount” is an amount sufficient, to achieve a desired biological effect, for example, an amount that is effective to decrease a symptom associated with a norovirus infection.

[0234] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent, such as a polypeptide conjugate, useful for reducing, alleviating and / or preventing a norovirus infection will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition, and the manner of administration of the therapeutic composition.

[0235] With respect to the aspects described herein, the term “subject” includes, but is not limited to, mammals, inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as pigs, cattle, sheep, horses) and companion animals (such as cats and dogs). In certain examples, the subject is a human. In other examples, the subject is a dog. In various examples, the subject is a bovine. In other examples, the subject is a pig.

[0236] As used herein, the terms “a disease, disorder or condition associated with norovirus” refer to diseases, disorders or conditions caused, directly or indirectly, by infection with a norovirus. This virus can be amplified and cause severe disease in domestic animals, such as pigs and cattle, norovirus may then be subsequently transmitted to humans (i.e., a zoonotic disease), where the infection is highly contagious and typically manifested as a severe gastrointestinal illness that may include nausea, vomiting, stomach pain or cramps, watery or loose diarrhoea, feeling ill, low-grade fever and myalgia.

[0237] Suitably, the present methods include the administration of a therapeutically effective amount of one or more further or additional therapeutic agents, such as an anti-inflammatory agent (e.g., NSAIDs, corticosteroids) and / or an antiviral agent (e.g., a milk oligosaccharide, NB-2, NB- 7, NB-45, NB-76, dasabuvir, nitazoxanide, remdesivir and nelfinavir), to the subject in question. As such, the polypeptide conjugate, composition, genetic construct, host cell or isolated nucleic acid described herein may be administered alone (i.e., monotherapy) or alternatively be administered in combination (e.g., concurrently, simultaneously, sequentially, successively or alternately) with the further therapeutic agent which aims to treat, ameliorate or prevent a noroviral infection or a disease, disorder or condition associated therewith. According to some examples, the present methods include the further administration of a therapeutically effective amount of a milk oligosaccharide, such as 2’ -FL, to the subject. In other examples, the present methods include the administration of a therapeutically effective amount of a further single domain antibody, such as NB-2, NB-7, NB-45 and NB-76 as described herein, to the subject.

[0238] Methods for diagnosing norovirus infections

[0239] The polypeptide conjugates, single domain antibodies and antigen binding molecules disclosed herein may be used to detect viral proteins or antigens, such as a VP1 or capsid protein of a norovirus, in vitro or in vivo. Such in vitro testing may involve obtaining a biological sample, such as faeces, saliva, vomit, mucous, blood, plasma or serum, from the subject. The detection of viral protein or elevated levels of viral protein in the biological sample from a subject may be indicative of a norovirus infection in said subject.

[0240] Accordingly, in one broad form, the present disclosure provides a method of diagnosing or monitoring a norovirus infection, and / or a disease, disorder or condition associated therewith, in a subject, said method including the step of contacting the subject and / or a biological sample from the subject with a polypeptide conjugate or a composition described herein.

[0241] In another form, the present disclosure provides a kit or device for diagnosing or monitoring a norovirus infection, and / or a disease, disorder or condition associated therewith, in a subject, said kit or device including the polypeptide conjugate or the composition described herein; optionally one or more reagents for detecting the polypeptide conjugate; and optionally instructions for use.

[0242] Such kits may include various diluents and buffers, labelled conjugates or other agents for performing the methods described above, and other signal-generating reagents, such as enzyme substrates, cofactors, chromogens, and fluorogens. Other components of the kit can easily be determined by one of skill in the art. Such components may include coating reagents, indicator charts for colorimetric and fluorometric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, a sample preparatory cup, etc. A kit may comprise buffers or other reagents appropriate for constituting a reaction medium in which the polypeptide conjugate described herein is contacted with the sample.

[0243] In another form, provided herein is a sensor or sensor device for detecting norovirus, comprising the polypeptide conjugate described herein.

[0244] The term “sensor” as used herein indicates a device that measures a physical quantity and converts it into a signal which can be read by an observer or by an instrument. As is understood by the skilled person, a sensor can be calibrated against known standards. Accordingly, a sensor can be used to capture a norovirus :polypeptide conjugate complex by exploiting the affinity of the polypeptide conjugate to the norovirus, and can be detected using techniques identifiable by a skilled person upon reading of the present disclosure.

[0245] In such examples of the present aspects, the polypeptide conjugate can be linked to, bound, affixed, associated with or otherwise coupled to a suitable substrate, as are well known in the art. To this end, the sensor may further comprise a substrate. The substrate may facilitate detection of the norovirus or a norovirus:polypeptide conjugate complex. The substrate may be any surface to which a polypeptide conjugate may be attached, directly or indirectly, through either covalent or non-covalent bonds. The substrate materials may be naturally occurring, synthetic, or a modification of a naturally occurring material. The substrate may be a bead, a matrix, a crosslinked polymer, a gel, a particle, a surface, a plate, a paper, a membrane, a well or other solid or semi-solid substrate, or any combination thereof.

[0246] Suitably, the substrate is a sensor chip surface (e.g., for BIACore or surface plasmon resonance), an ELISA / ELLBA plate, a sepharose, an agarose, Protein A, Protein G, a magnetic bead, a paramagnetic particle, nanomaterials, such as, a nanoparticle, an atomic cluster, a dot (e.g., quantum dots or carbon dots) or any other substrate known to those skilled in the art.

[0247] Suitably, such kits may further include a detectable probe, as are known in the art, for binding the polypeptide conjugate, such as in the form of a norovirus:polypeptide conjugate complex. Accordingly, the present methods may further include the step of contacting the subject and / or the biological sample from the subject with a detectable probe for binding a norovirus:polypeptide conjugate complex. In other examples, the polypeptide conjugate may be linked, coupled or otherwise associated with a detectable probe. In some examples, the probe comprises a metal selected from the group consisting of gold, silver, titanium, stainless steel, aluminium, platinum, alloys thereof, and / or combinations thereof. In certain examples, a probe comprising nanoparticles (e.g., gold nanoparticles) is provided.

[0248] In another form, provided herein is a method of producing a sensor for detecting norovirus, said method including the steps of:

[0249] (a) providing a substrate; and

[0250] (b) coupling, binding, affixing or otherwise linking the polypeptide conjugate described herein to the substrate, to thereby produce the sensor.

[0251] In yet another form, provided herein is a method of detecting norovirus in a sample, said method including the steps of:

[0252] (a) contacting the sample with the polypeptide conjugate described herein or the sensor described herein; and

[0253] (b) detecting the presence or absence of or measuring the level of a norovirus:polypeptide conjugate complex, to thereby detect norovirus in the sample.

[0254] In a further form, provided herein is a method of detecting a norovirus infection in a subject, said method including the steps:

[0255] (a) contacting a biological sample obtained from the subject with the polypeptide conjugate described herein or the sensor described herein; and

[0256] (b) detecting the presence or absence of or measuring the level of a norovirus:polypeptide conjugate complex, to thereby detect the norovirus infection in the subject.

[0257] Methods of “detecting” or “determining a level of’ or “measuring the level of’ binding are not particularly limited so long as they can detect binding between the polypeptide conjugate described herein and the norovirus present in the sample. Examples include surface plasmon resonance, high resolution microscopy, such as electron microscopy or confocal microscopy and immunosorbent assays, in which the norovirus and / or norovirus :polypeptide conjugate complex can be detected, and optionally quantified. It is contemplated that such levels of binding of the polypeptide conjugate to the norovirus (e.g., levels of norovirus:polypeptide conjugate complexes) can be directly correlated to a level or concentration of norovirus in the sample, as per standard methods known in the art.

[0258] It is considered that terms such as “contacting”, “exposing” or “applying” are terms that can, in context, be used interchangeably in the present disclosure. The term contacting requires that the polypeptide conjugate be brought into contact with a sample so as to form detectable complexes (e.g., a norovirus :polypeptide conjugate complex). Such binding may be detected using various techniques known in the art. For example, a surface plasmon resonance sensor chip or device including the polypeptide conjugate described herein may be used. An immunoassay may also be used incorporating the polypeptide conjugate described herein.

[0259] It is envisaged that determining conditions that enable binding of anpolypeptide conjugate described herein to a norovirus would be well within the purview of those skilled in the art. In general, a polypeptide conjugate of the disclosure can be provided in a suitable solution and concentration so that they may recognise and bind to the norvirus in a sample.

[0260] Some methods of detecting norovirus comprise contacting a sample with a polypeptide conjugate described herein, and detecting the presence or absence of or measuring a level of the norovirus and / or norovirus:polypeptide conjugate complex in the sample. Detection of the norovirus:polypeptide conjugate complex may indicate the presence of the norovirus. No detection of the norovirus :polypeptide conjugate complex may indicate the absence of norovirus. Measuring the level of the norovirus and / or the norovirus :polypep tide conjugate complex in the sample can provide a quantitative determination or indication of the number or concentration of norovirus in a sample.

[0261] A sample may be contacted with an polypeptide conjugate described herein under conditions and for an amount of time sufficient to permit the polypeptide conjugate to bind to a norovirus and form a norovirus:polypeptide conjugate complex. For example, a sample can be incubated with a polypeptide conjugate. The incubation may be performed at room temperature (e.g., 25°C). Alternatively, the incubation may be performed at 4°C. Alternatively, the incubation may be performed at higher temperature (e.g., including up to and at the melting temperature of the polypeptide conjugate). Alternatively, the incubation may be performed at a temperature higher than the melting temperature of the polypeptide conjugate (e.g., up to 90°C). The incubation may be performed overnight. Alternatively, the incubation may be performed for at least about 1 minute (e.g., at least about 1, 2, 5, 10, 20, 30, 40, 50, 60 minutes or any range therein), at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour (e.g., at least about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 24 hours or any range therein), at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, or at least about 24 hours. Alternatively, the incubation may be performed for no more than 1 hour, no more than 45 minutes, no more than 30 minutes, no more than 15 minutes, or no more than 10 minutes. A precise incubation time may not be required, and norovirus detection may be performed immediately after contacting the polypeptide conjugate with the norovirus.

[0262] Detection may include methods comprising direct labelling of a polypeptide conjugate (e.g., with a modified nucleotide, labelled nucleotide or tag incorporated into the polypeptide conjugate) or merely by binding of the polypeptide conjugate with the norovirus thereby forming a norovirus:polypeptide conjugate complex. For example, the polypeptide conjugate may be detectably labelled, such as at a 5' end and / or 3' end thereof, or capable of binding a detectable label, such as those hereinafter described. The polypeptide conjugate may be linked to an enzyme, enzyme substrate, a fluorescent or fluorescent substrate, chemiluminescent molecule, chemiluminescent substrate, purification tag and / or a solid support. In one example, the norovirus:polypeptide conjugate complex may be directly or indirectly detected.

[0263] The present methods may include the step of determining a presence or absence of a norovirus infection in said subject, wherein the presence of a viral protein, such as an VP1 protein, of a norovirus, indicates a current or previous norovirus infection. As such, the subject and / or a biological sample therefrom may be contacted with the polypeptide conjugate or the composition provided herein for a time and under conditions sufficient to detect antigen- specific binding thereof. Suitably, the method of this aspect is for determining a relative or absolute level of the viral protein in the biological sample.

[0264] The present method may further include the earlier step of collecting the biological sample from the subject. Such a sample may be obtained by freshly collecting a sample, or may be obtained from a previously collected and stored sample. By way of example, a sample may be obtained from a previously collected and stored (e.g., refrigerated or frozen) stool or vomit sample. Suitably, a sample is obtained by freshly collecting a sample from the subject. Alternatively, a sample can be obtained from a previously collected and stored sample from the subject. Once collected the sample may be processed in a way, such as purifying, concentrating or solubilising, to make it more suitable for the subsequent contacting and / or detection steps (e.g., concentration, isolation or purification of one or more virus particles or proteins within the biological sample). Such assays may include immunoassays, such as western blot and ELISA. It should be understood, however, that this disclosure is not limited by reference to the specific methods of detection or immunoassays disclosed herein.

[0265] In some examples, the methods described herein may be performed in a lateral flow assay (LFA) format or the device or kit may comprise an LFA device. LFAs, also known as “immunochromatographic strip tests”, have been a popular platform for rapid immunoassays since their introduction in the mid-1980s. LFAs are particularly suitable where a rapid test is required or where specialized laboratory equipment is not available. In hospitals, clinics, physician offices, and clinical laboratories, LF-based tests are used for the qualitative and quantitative detection of the presence of a specific analyte in a liquid sample. LFAs operate on the same principles as ELISA. In essence, these tests run a liquid sample along the surface of a membrane or filter paper with reactive molecules that show a visual positive or negative result depending on the presence of a particular analyte. A LFA device, is a device configured to receive a sample at a sample region and to provide for the sample to move laterally, via, e.g., wicking, by capillary action from the sample region to a detection region.

[0266] The methods of the present disclosure can be performed on various biological samples. As used herein, the term “biological sample” is suitably a sample obtained from a subject. For example, the biological sample can be a bodily fluid of the subject. In certain examples, the biological sample is selected from a group consisting of blood, serum, plasma, urine, saliva, faeces, tears, vomit, broncho-alveolar lavage fluid (BALF), cerebrospinal fluid (CSF), sweat and seminal fluid. In certain examples, the biological sample is a faecal or stool sample. In other examples, the biological sample is a vomit sample. In some examples, the biological sample is a mucous sample. For particular examples, the biological sample is a saliva sample. In another example, the biological sample may be from a rectal swab comprising rectal fluids.

[0267] In another form, the present disclosure provides a method of isolating or purifying a norovirus from a sample, such as a biological sample, said method including the steps of:

[0268] (a) contacting the sample with the polypeptide conjugate described herein; and

[0269] (b) isolating a norovirus:polypeptide conjugate complex from the sample, to thereby isolate or purify a norovirus from the sample.

[0270] Methods for isolating or purifying a norovirus or an immune complex comprising same from a sample will be apparent to the skilled person and / or are described herein.

[0271] In this context, the term “isolating” suitably refers to at least partly purifying, concentrating or removing the norovirus from or within the sample. Affinity based separation methods may be used. A tag or label on a polypeptide conjugate described herein may also be used so that the norovirus can be filtered or sorted from a sample, such as by a fluorescence- or affinity chromatography-based sorting system.

[0272] Suitably, the polypeptide conjugates for use in the present methods are labelled, such as by way of conjugation to a detectable or functional label or marker. Various methods of labelling proteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionucleotides (such as35S,nC,13N,150,18F,19F, "TC,131I,3H,14C,15N,90Y, "TC,i nIn and125I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, betagalactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents (such as gadolinium chelates). In some examples, labels are conjugated or operably linked to the polypeptide conjugate by one or more linkers of various lengths to reduce potential steric hindrance for the binding of the single domain antibody or antigen binding molecules disclosed herein to a viral protein.

[0273] Suitably, the present methods further include the step of detecting and / or measuring a level of antigen binding to the polypeptide conjugate. Any method or assay known in the field can be employed in the diagnostic and prognostic methods of the invention, e.g., ligand binding assays, immunoassays, competition binding assays, etc., for detecting and / or measuring antigen determining the presence of a norovirus, or a viral particle, protein or antigen derived therefrom.

[0274] Suitably, the diagnostic methods described herein may include the step of administering a treatment to the subject. By way of example, this can include administering to the subject a therapeutically effective amount of a treatment, such as those therapeutic agents, inclusive of the polypeptide conjugate or composition, described herein, when one or more noroviral proteins or antigens (e.g., VP1) are detected in the subject or a biological sample obtained therefrom (e.g., method is diagnostic or indicative of the subject having a noroviral infection). More particularly, this may include administering to the subject a therapeutically effective amount of an anti-viral agent, such as a polypeptide conjugate or composition described herein, when the method is diagnostic or indicative of the subject having a noroviral infection.

[0275] It is envisaged that the polypeptide conjugate disclosed herein can be used in vitro and in vivo to monitor the course of a norovirus infection, such as during therapy thereof. Thus, for example, by measuring the increase or decrease in a level of a viral particle / protein / antigen or the number of cells infected with a norovirus or changes in the subject or in a biological sample therefrom, it would be possible to determine whether a particular therapeutic regimen aimed at treating or ameliorating a norovirus infection, and / or a disease, disorder or condition associated therewith, is effective.

[0276] Nucleic acid delivery

[0277] The present disclosure also provides certain aspects relating to the administration of one or more nucleic acids encoding the polypeptide conjugates described herein (e.g., a polypeptide conjugate comprising, consisting essentially of or consisting of an amino acid sequence set forth in any of SEQ ID NOs: 12 to 15; or a nucleotide comprising, consisting essentially of or consisting of a nucleic acid sequence set forth in any of SEQ ID NOs: 17 to 20), or fragments, variants or derivatives thereof, for:

[0278] (i) passively immunizing a subject against a norovirus infection; and / or

[0279] (ii) treating, ameliorating or preventing a norovirus infection in a subject.

[0280] Accordingly, in one particular form, there is provided herein a method of treating and / or preventing a norovirus infection, and / or a disease, disorder or condition associated therewith in a subject, said method including the step of administering to the subject a therapeutically effective amount of an isolated nucleic acid encoding a polypeptide conjugate provided herein.

[0281] In some examples, the isolated nucleic acid encoding the polypeptide conjugate is in the form of a genetic construct suitable for administration to a mammal, such as a human, inclusive of those hereinbefore described. More particularly, the genetic construct may be suitable for DNA delivery of the polypeptide conjugates to a mammal such as a human. A useful reference describing DNA delivery of peptides is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006).

[0282] According to certain examples, the isolated nucleic acid encoding the polypeptide conjugate is in the form of RNA, such as mRNA, suitable for administration to a mammal, such as a human. In various examples, the isolated nucleic acid is or comprises an mRNA having an open reading frame encoding a polypeptide conjugate provided herein. mRNA vaccines are described, for example, in International Patent Application Nos. PCT / US2015 / 027400 and PCT / US2016 / 044918, herein incorporated by reference in their entirety. The mRNA delivery of antibodies, such as single domain antibodies, is further described in PCT / US2018 / 037918, which is also incorporated by reference herein.

[0283] It will be appreciated that nucleic acid, and more particularly mRNA, delivery of single domain antibodies (and polypeptide conjugates comprising such single domain antibodies) provides a unique therapeutic alternative to peptide-based or DNA-based methods of administering such agents. When the mRNA is delivered to a cell, the mRNA will be processed into a polypeptide or peptide by the intracellular machinery which can then process the polypeptide or peptide into the polypeptide conjugates capable of binding a viral protein or antigen of a norovirus on or in a virally-infected cell or biological fluid of the subject.

[0284] Compositions comprising isolated nucleic acids or polynucleotides that encode the polypeptide conjugates described herein, or fragments, variants or derivatives thereof that may be used for such methods, are also contemplated by the present disclosure. For such examples, the composition suitably comprises a delivery agent or system, such as a nanoparticle, a lipid nanoparticle or a liposome, as are known in the art. In various examples, the nanoparticle has a mean diameter of 50-200 nm. In some examples, the composition comprising the mRNA polynucleotide (e.g., an mRNA polynucleotide having an open reading frame that encodes a polypeptide conjugate described herein) is formulated in a lipid nanoparticle.

[0285] Methods of inactivating norovirus in vitro

[0286] The present disclosure further envisages use of the polypeptide conjugates described herein for decontaminating or disinfecting, for example, surfaces or substrates that may be contaminated with a norovirus. To this end, it is well known that norovirus can be spread indirectly by way of human contact with such contaminated substrates and surfaces, such as contaminated body surfaces, environmental surfaces, food and water. In this regard, the polypeptide conjugates described herein may be essentially functional as a disinfectant agent or an antiviral agent.

[0287] Accordingly, in one form, the present disclosure provides a method for inactivating or neutralising a norovirus, such as a virus particle thereof, associated with a substrate or surface (e.g., in and / or on the substrate or surface), said method including the step of contacting the substrate with an effective amount of the polypeptide conjugate or the composition provided herein to thereby inactivate or neutralise the norovirus associated with the substrate or surface.

[0288] In particular examples, the surface or substrate is a body surface, such as of a subject currently infected with a norovirus or a subject in contact with such an infected subject.

[0289] In other examples, the surface or substrate is an environmental surface or substrate.

[0290] In view of the foregoing, the composition provided herein may be considered a disinfectant composition for inactivating a norovirus associated with a substrate or surface. Such disinfectant compositions may comprise one or more additional components with disinfectant properties in relation to viruses, such as an alcohol, a protease, an RNase, a detergent and a disinfectant (e.g., sodium hypochlorite).

[0291] So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples.

[0292] Examples

[0293] Example 1

[0294] This Example studied the structural and functional properties of a modified NB26 nanobody. Native NB26 using an IgFc (termed Fc-NB26) was dimerized and the neutralization capacities of both native NB26 and Fc-NB26 were examined using a human intestinal enteroid (HIE) culture system. Furthermore, to accurately define GII antigenic variation, the X-ray crystallography structures of GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1 P domains were determined and the binding with Fc-NB26 to these new structures was determined using a direct EEISA. The functional properties of a modified NB85 nanobody were also examined. Native NB85 was conjugated to an IgFc (termed Fc-NB85) and dimerized. The binding characteristics of Fc-NB85 to these new structures was determined using a direct ELISA.

[0295] Materials and Methods

[0296] Sequence analysis. The full-length capsid amino acid sequences of GII.4c (2), GII.4-CHDC (ACT76142, CHDC-1974), GII.4-Syd (JX459908, Syd-2012), GII.8 (AF195848, Amsterdam), GII.10 (AF504671, Vietnam026), GII.14 (AY130761, M7), GII.17-CS-E1 (AY502009, CS- El), GII.24 (KY225989, Loretol972), GII.26 (KU306738, Leon4509), and GII.NA1 (NA: Not Assigned, MG495079, Loreto 1257) were aligned using Clustal Omega. These capsids had 64-91% amino acid identity and varying lengths, GII.8 (537 amino acids), GII.14 (536 amino acids), GIL 17 (539 amino acids), GII.24 (542 amino acids), GII.26 (542 amino acids), and GII.NA1 (542 amino acids).

[0297] Norovirus P domain and VLP production. The norovirus P domain gene of GII.4c, GII.8, GII.14, GIL 17, GII.24, GII.26, and GII.NA1 were cloned and expressed. Briefly, the P domain was cloned into a modified expression vector (pMal-c2X) and transformed into E. coli BL21 cells, which were grown in LB medium at 37 °C. Expression was induced with 0.7 mM IPTG (OD600 = 0.6) for 18 h at 22 °C. Cells were harvested by centrifugation and disrupted by sonication on ice. The His-tagged fusion-P domain protein was purified from a Ni-column (Qiagen) and digested with HRV-3C protease (Novagen) overnight at 4 °C. The cleaved P domain was separated on the Ni-column and dialyzed in gel filtration buffer (25 mM Tris-HCl (pH 7.6) and 300 mM NaCl) overnight at 4 °C. The P domain was further purified by size exclusion chromatography (SEC) using a AKTA Prime with a Superdex 75 / 200 column, concentrated to 2-4 mg / mL, and stored at 4 °C. The GII.4 and GII.10 VLPs were produced as described (3).

[0298] Fc-NB26 and Fc-NB85 design and production. NB26 and NB85 were expressed in E. coli WK6 cells as described (4). To generate Fc-NB26 and Fc-NB85, the C-terminus of NB26 or NB85 was designed to contain the pINFUSE-hlgGl-Fcl sequence (pINFUSE-hlgGl-Fcl, InvivoGen) without introns, while the N- and C-terminus contained ATTB1 and ATTB2 sequences, respectively. The Fc-NB26 and Fc-NB85 sequence were commercially synthesized (Invitrogen) for Gateway cloning (pDEST8) and expression in the insect cell baculovirus system. Briefly, bacmids were generated from the pDEST-Fc-NB26 and pDEST-Fc-NB85 plasmid, for transfection of Sf9 cells. After 5 days post transfection of Sf9 cells, the culture was centrifuged at 1057 x g for 10 min at 4 °C (to develop seed Fc-NB26 and Fc-NB85 recombinant baculovirus). The Fc-NB26 and Fc-NB85 seed virus was subsequently used to infect HighFive insect cells (Invitrogen). After 4-5 days post infection (dpi), the Fc-NB26 and Fc-NB85 was separated from insect cells at 4,960 x g for 1 h at 4 °C. The Fc-NB26 and Fc-NB85 containing supernatant was mixed 1:1 with Protein A IgG Binding Buffer (Pierce), incubated on the Protein A Agarose (Pierce), and then eluted using Protein A Elution Buffer (Pierce). Approximately 20 mg / L of purified Fc-NB26 and Fc-NB85 was prepared and was stable at 4 °C for >6 months. Fc-NB26 and Fc-NB85 expression was confirmed using SDS-PAGE and SEC, concentrated to 0.6 mg / mL, and stored at 4 °C.

[0299] Cross-reactivity studies using ELISA. The cross-reactivity of Fc-NB26 and Fc-NB85 were determined using a direct ELISA as previously described with slight modifications (5, 6). Microtiter plates (MaxiSorp, ThermoFisher) were coated with 100 pL / well (10 pg / mL) of P domain overnight at 4 °C. Plates were washed three times with phosphate -buffered saline (PBS; pH 7.4) containing 0.1% Tween 20 (PBS-T) and subsequently blocked with 5% skimmed milk in PBS for 1 h at room temperature. Fc-NB26 or Fc-NB85 was (1:1) serially diluted in PBS (starting at 1.2 pg / mL) and 100 pL / well applied to the washed microtiter plates and then incubated for 1 h at 37 °C. The plates were washed and 100 pL of 1:4,000 goat anti-human-Fc HRP-conjugated Mab was added to each well (#31413, ThermoFisher). Plates were washed and then developed with 50 pL of TMB (OptEIA TMB Substrate Reagent Set, BD) in the dark for 30 min at room temperature. Finally, the reaction was stopped with 25 pL of 6% (v / v) HC1 and absorption was measured at optical density 450 nm (OD450). For this assay, a cutoff limit was set at OD450 > 0.18, which was ~3 times the value of the PBS negative control (OD450 = 0.06). A negative control rabbit haemorrhagic disease virus P domain showed no cross -reactivity with Fc-NB26 or Fc-NB85 (data not shown).

[0300] Isothermal titration calorimeter (ITC) measurements. A Nano ITC (TA instruments) with a cell volume of 170 pL was used to determine the binding affinity of NB26 and Fc-NB26 to GIL 10 and GII.4-Syd P domains. Each experiment was performed in duplicate, and heat of NB26 titration into PBS (pH 7.4) was subtracted for each trace. A concentration of 100 pM of NB26 was titrated as 30 injections of 1.5 pL every 200 seconds, into the cell containing between 10-12 pM of P domain. For Fc-NB26, 30 injections of 30 pM of Fc-NB26 were titrated into 3 pM of P domain. The baseline was equilibrated for 600-900 seconds before the first injection. Statistics were performed using a paired Student’s T-test with a confidence level of 95-99%. Crystallization of norovirus P domains. P domains crystals were grown using the hanging drop method and the following mother solutions, GII.4c (0.2 M magnesium formate dihydrate and 20% w / v PEG3350), GII.8 (0.2 M sodium chloride, 0.1 M sodium potassium phosphate (pH 6.2) and 50% v / v PEG200), GIL 14 (0.1 M bicine and 10% v / v MPD), GIL 17 (0.2 M TMAO, 0.1 M Tris (pH 8.5), and 50% w / v PEG2000 MME), GII.24 (0.2 M lithium sulfate, 0.1 M Bis-Tris (pH 5.5), and 25% w / v PEG3350), GII.26 (0.1 M ammonium acetate, 0.1 M Bis-Tris (pH 5.5), and 17% w / v PEG10,000), and GII.NA1 (0.2 M magnesium chloride hexahydrate, 0.1 M Tris (pH 7.0), and 10% w / v PEG8000) for 6-10 days at 18 °C. Prior to data collection, crystals were transferred to a cryoprotectant containing the mother solution in 30% ethylene glycol, followed by flash freezing in liquid nitrogen. For the GII.4c P domain HBGA complexes and GII.4-Syd and GIL 10 2’-FL complexes, the present inventors either soaked a 60-molar excess of HBGA or 2’ -FL into premade crystals and / or co-crystallized, respectively (7-9). Prior to data collection, crystals were transferred to a cryoprotectant containing the mother liquor, 30% ethylene glycol, and 30-60-molar excess of HBGA or 2’ -FL.

[0301] Data collection, structure solution, and refinement. X-ray diffraction data were collected at MX1 and MX2 beamlines at the Australian Synchrotron, Australia and processed with XDS or AIMLESS (9, 10). Structures were solved by molecular replacement in PHASER Phaser-MR (11) using either GII.4-Syd (PDB ID: 4OOS) or GIL 10 (PDB ID: 3ONU) P domains as search models. Structures were refined in multiple rounds of manual model building in COOT (12) and PHENIX (13). Structures were validated with Procheck (.14) and Molprobity (15). Protein interactions were analyzed using PyMOL Molecular Graphics System, Version 2.1 Schrodinger, LLC (1.6). Atomic coordinates and structure factors are deposited in the Protein Databank.

[0302] Analysis of GII.4 capsid sequence conservation. Amino acid sequence conservation was analyzed for GII.4 variants using an alignment over 2000 GII.4 capsid sequences. Residue conservation was computed using the AL2CO server with a Henikoff-Henikoff sequence weighting scheme, normalized conservation values, and entropy-based conservation calculation (17). The computed residue conservation scores were mapped and colored white (highly variable) to deep purple (highly conserved) onto the surface of the unbound GII.4-Syd (PDB ID: 4OOS) structure using AL2CO (17) and a PyMOL script. Binding interfaces and interactions were analyzed using PDBePISA online server (https: / / www.ebi.ac.uk / pdbe / pisa / ) (18) and PyMOL (version 1.2), with hydrogen bond distances -2.36-3.88 A and electrostatic distances -2.56-3.89 A. Water-mediated interactions were excluded from the analysis. HIE culture system and NB26 and Fc-NB26 inhibition studies. Secre tor-positive jejunal HIE culture (J2) and Noggin producing cell lines were provided by Baylor College of Medicine, USA and processed as described with slight modifications (19) using a positive GII.4 norovirus stool sample (19). Briefly, HIE were trypsinized and grown as 2D monolayers in a collagen coated 96 well plates. After one day, the complete media growth factor positive (CMGF+) media was changed to differentiation media for five days. Monolayers were pretreated with 500 pM glycochenodeoxy cholic acid (GCDCA) for two days before infection experiments. Confluent monolayers were washed once with ice cold CMGF- and incubated for 1 h at 37 °C with diluted stool samples. After infection, monolayers were washed twice with ice cold PBS and incubated in differentiation media supplemented with 500 pM GCDCA or 500 pM GCDCA and 1% human bile. Samples were frozen at 0 days post infection (dpi), i.e., 1 h after virus attachment and 3 dpi. For inhibition experiments, diluted stool samples were preincubated with serially diluted NB26 or Fc-NB26 for 1 h at 37 °C. The concentrations of NB26 and Fc-NB26 were calculated using a Nanodrop (ThermoFisher). For Fc-NB26, the concentration was calculated using a extinction coefficient at 280 nm for the Fc-NB26 amino acid sequence of 1.492 (20). Isotype IgG (antimyosin heavy chain antibody, Abeam) at 200 pg / mE was used as a negative control. Confluent monolayers were washed once with ice cold CMGF- and incubated for 1 h at 37 °C with diluted stool samples. After infection, monolayers were washed twice with ice cold PBS and incubated in differentiation media. Plates were frozen at 0 dpi and 3 dpi. Viral RNA was extracted using RNAeasy mini kit (Qiagen) or phenol / chloroform extraction with RiboZol according to manufacturer instructions. Genome copy levels were measured using qScript XLT One-step RT- qPCR ToughMix reagent with ROX (Quanta Biosciences) using COG2R / QNIF2D primer pair, and probe QNIFS as described previously. All experiments were performed three times with technical quadruplicates. A standard curve based on human norovirus RNA transcript was used to quantitate viral genome equivalents. Percent inhibition in viral genome equivalents relative to the IgG control was determined. Differences were considered significant when P value < 0.05. The ICso values were analyzed using Microsoft Excel and GraphPad Prism (version 10.0).

[0303] Negative stain electron microscopy of untreated and Nanobody treated VLPs. Untreated GII.10 VEPs were diluted to 0.03 mg / mL in TBS (10 mM Tris HC1 and 150 mM NaCl (pH 7.5)) and stained on carbon-coated Ted Pella grids using 2% uranyl formate. For the complexes, GIE10 VLPs were treated by mixing 1.0 mg / mL GII.10 VLP with 1.0 mg / mL of NB26 or Fc-NB26 for 1 h at room temperature. The mixtures were then diluted to 0.05 mg / mL with distilled water before staining on carbon-coated Ted Pella grids using 2% uranyl formate. Each grid was analyzed on a Talos (L120C) TEM and images collected at 92,000 > magnification. Results

[0304] A conserved dynamic HBGA pocket is observed for many GII genotypes. The X-ray crystal structures of GII.8, GII.14, GII.17-variant, GII.24, GII.26, and a non-assigned genotype (GII.NA1) P domains were solved to 1.27-1.74 A resolution. (Table 1). Norovirus P domains expressed in E. coli typically form a biologically relevant homodimer (8) and like other structures, these six Pl subdomains are comprised of eight P-strands and one a-helix, while the P2 subdomains contain six P-strands (Fig. 1A). These six P domains were structurally equivalent with a root mean square deviation (RMSD) for C-alpha atoms of 0.67-0.94 A. The principal structural variation among the models is a variable length P2 subdomain loop, which was termed Loop A (chain A residues -337- 354). Loop A is 5-7 residues longer in GII.24, GII.26 and GII.NA1 P domains compared to GII.8, GIL 14, and GIL 17 P domains (Fig. 1A and Table 2). An extended Loop A is also present in GII.2, GII.6, and GII.10, and GIL 19 P domains, whereas a shortened Loop A is found in GII.l, GII.3, GII.4, GII.l 1, and GII.12 P domains (Table 2). The X-ray crystal structures of these six P domains in complex with HBGAs is not yet determined, but superposition with a GII.10 P domain A-type trisaccharide complex structure revealed a conserved set of residues capable of interacting with the fucose moiety of HBGAs. These include side- (underlined) and main-chain interactions for GII.8 (residues T346, R347, D375, and G440), GIL 14 (residues T345, R346, D374, and G439), GIL 17 (residues T349, R350, D378, and G443), GII.24 (residues N354, R355, D384, and G446), GII.26 (residues N353, R354, D383, and G445), and GII.NA1 (residues N354, R355, D384, and G446) (Fig. IB).

[0305] Structural analysis of other GII X-ray crystal structures shows that, in at least 15 genotypes and closely matching genetic variants, conserved fucose binding residues are present (8, 9, 21 ). Present analysis shows the HBGA binding pocket is formed by a quaternary interaction at the P domain dimeric interface involving loops from chains A and B. Loop A (chain A residues -337-354), described above, together with Loop B (chain A residues -371-391), Loop D (chain A residues -390-407), and Loop C (chain B residues -434-454) form the main HBGA pocket (Fig. 1 and Table 2). At its base, Loop A contains a conserved arginine residue whose side chain binds the fucose moiety of HBGAs. Loop B has a highly conserved, well-positioned aspartic acid, which forms two direct hydrogen bonds with the fucose moiety of HBGAs. Loop C has one main chain residue that almost always forms a direct hydrogen bond with the fucose moiety (Figs. IB and 1C) (8, 9). The placement of Loops B and C are also conserved in these novel structures, whereas Loop D is more variable in length among the genotypes. Also, Loop D contains a few variable residues that loosely bind (e.g., through water mediated interaction) HBGA saccharides other than fucose (5, 8, 9). Overall, the dynamics of the HBGA binding pocket is mainly formed by quaternary interactions, with three main loops, Loops A and B (chain A) and Loop C (chain B) strongly holding the fucose. The amino acid variation surrounding the HBGA pocket might select for different HBGA types among the human population (9, 21, 22), thus increasing norovirus infection rates by binding to multiple HBGA types while at the same time escaping from host immune responses. Of note, two P domains in this analysis (GIL 11 and GIL 19) were isolated from pigs (Table 2), stressing that these important structural features are shared with animal noroviruses.

[0306] The engineered GII.4c VLP introduces variability near the HBGA pocket. To precisely compare the engineered GII.4c immunogen with natural genetic diversity, the first X-ray crystallographic structure of the GII.4c P domain was solved at 1.34 A resolution (Fig. 2A and Table 3). Like all other native GII.4 P domains, the GII.4c P domain is subdivided into the Pl subdomain (residues 225-281 and 421-530) with eight P-strands and one a-helix and P2 subdomain (residues 282-420) with six P-strands. Nine of thirteen amino acid substitutions in GII.4c are exposed on the surface of the P2 subdomain and several residues (S372, F375, and S393) sit in the HBGA pocket (Fig. 2B). To better understand if these amino acid substitutions affected HBGA binding interactions at the atomic level, the present inventors determined the X-ray crystallographic structure of the GII.4c P domain in complex with H2-type at 1.61 A resolution (Fig. 2A and Table 3). Unfortunately, the GII.4c P domain did not form complexes with any other HBGA types, regardless of soaking and co -crystallization conditions. Nevertheless, the GII.4c P domain interacts with the fucose moiety of H2-type through a set of highly conserved residues (R345, T344, D374, and Y444), which were not substituted in the engineered immunogen (Fig. 2B). Comparing the GII.4c substitutions with an amino acid diversity plot of GII.4 genetic variants showed that many substitutions are in variable patches on the P2 subdomain surface (Figs. 2C and 2D). When the amino acid diversity is mapped using one representative from each of the 29 genotypes, most of the top surface of the P2 subdomain is highly variable, except for the two HBGA pockets and the lateral region between the HBGA pockets (Fig. 2E). It was previously shown that this conserved region binds two additional fucose subunits on the GIL 10 P domain (Fig. 2E), although the function(s) of the fucose binding sites remains unclear (23). Overall, the present extensive structural analysis of 15 genotypes supports the notion that this HBGA pocket is relatively conserved, making it an attractive universal norovirus infection therapeutic target.

[0307] An engineered Fc-linked Nanobody (Fc-NB26). To convert the broadly reactive NB26 into a next generation therapeutic, the C-terminus of the Nanobody was linked onto a human Fc sequence and expressed the modified Nanobody (termed Fc-NB26) in insect cells (Fig. 3). A direct ELISA was used to evaluate the cross -reactive binding capacity of Fc-NB26 with numerous GII genotypes (GII.4, GII.8, GII.10, GII.14, GII.17, GII.24, GII.28, and GII.NA1) (Fig. 4). Fc-NB26 bound to these eight genotypes, including the GII.4 variants, at concentrations ranging from 5-78 ng / mL, equivalent to NB26 which detected GII.l, GII.2, GII.4, GII.10, and GII.17 VLPs (4, 24). Similarly, Fc-NB85 bound to these eight genotypes, including the GII.4 variants, at comparable concentrations as Fc-NB85 (Fig. 4),

[0308] Superposition of the structure of a GII.10 P domain bound to NB26 and NB85 with GII.8, GII.14, GII.17, GII.24, GII.28, GII.NA1 P domain structures revealed no steric clashes with NB26 (Figs. 5A and 5B). Interestingly, a recently described neutralizing Nanobody (termed M4) bound at a site overlapping the NB26 epitope and shared several equivalent hydrogen bond interactions in this cavity (3, 4, 25). Previous studies have showed that NB26 blocked GII.4 and GII.10 VLPs from binding to HBGAs in a surrogate HBGA blocking assay (4). To further examine the therapeutic potential of NB26 and Fc-NB26, neutralization using the norovirus HIE culture system was analyzed with a GII.4 norovirus (Fig. 5C). It was found that NB26 neutralized norovirus replication with an IC50 value of 104.6 ng / mL (-7.0 nM), whereas Fc-NB26 neutralized with an IC50 value of 15.5 ng / mL (~1.0 nM) (Fig. 5D). The NB26 and Fc-NB26 neutralization results were comparable to the M4 Nanobody with IC50 values ranging between 53-379 ng / mL (4-25 nM) for different GII.4 variants (25). Similarly, a GII.4 specific IgG Mab (termed 10E9) neutralized a GII.4 variant with an IC50 value of 97 ng / mL (~0.6 nM) (.19). Likewise for GV murine norovirus, the present inventors previously identified several Nanobodies (NB5820 and NB5829) that neutralized murine norovirus in cell culture with IC50 values between 40-900 ng / mL (2-60 nM) (26). Finally, to better comprehend the neutralization mechanism of Fc-NB26, the present inventors examined untreated, NB26 treated, and Fc-NB26 treated GIL 10 VLPs using negative stain electron microscopy. Untreated GII.10 VLPs had a similar morphology to NB26 treated GIL 10 VLPs, whereas Fc-NB26 treated GIL 10 VLPs clearly resulted in particle disassembly and aggregation (Fig. 5E).

[0309] The binding affinities of Fc-NB26 and NB26 to GII.4-Syd and GIL 10 P domains were measured using ITC (Fig. 6). The affinities of NB26 to GII.4-Syd and GIL 10 were similar, with Kd values of 441 nM and 427 nM, respectively. As expected for a dimeric molecule, the affinities of Fc- NB26 to GII.4-Syd and GII.10 P domains were significantly higher than NB26, with Kd values of 5.87 nM and 0.82 nM, respectively, and a stoichiometric ratio of ~2:1 (Students’ T-test, P value < 0.05). Conclusions

[0310] The present Example showed that the norovirus GII HBGA binding pocket uses a regular set of 3 to 5 fucose binding residues. The extensive amino acid variation just beyond and surrounding this small set fucose binding residues is likely an evolutionary mechanism to avoid immune surveillance and a shared strategy among the GII genotypes. The present inventors previously screened Nanobody libraries developed against GII.4, GII.10, and GII.17 VLPs and discovered four Nanobodies that directly bound at the HBGA pockets but were genotype specific (3, 4, 10). Similarly, mAbs with neutralization capacities that bind on the top of the P2 subdomain and even the side of the P domain are mainly genotype specific (19, 27, 28).

[0311] It was found that NB26 modifications into an Fc-linked Nanobody improved binding affinities and increased neutralization capacities. The structural data and sequence alignments (Fig. 7) herein suggests that Fc-NB26 and Fc-NB85 could bind most GII genotypes. Development of such broadly reactive potent therapeutic norovirus Nanobodies (Figs. 8 and 9) delivered as a slow releasing prophylactic would be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

[0312] Table 1. Data collection and refinement statistics for six GII genotype crystal structures

[0313] GII.8 Amsterdam G9.14 M7 GII.17 CS-Et

[0314] Data co! lection

[0315] Space group P2?2,2, C121 P12^1

[0316] Cell dimensions af8, c (A) 67.23, 78.43. 115.39 105.82. 91.83, 67.11 82.33. 72.03, 81 .87

[0317] Protein 4718 4711 4787

[0318] Water 723 587 860

[0319] Ligand W 30 87

[0320] B-factors (A*) 25.07 23.23 1S.12

[0321] Protein 23.98 22.43 13-30

[0322] Water 32.09 29.52 24.88

[0323] Ligand 46.84 28.43 25.51

[0324] RMS bend length (A) 0.006 0,008 0.005

[0325] RMS bond angle f) 0.88 1.96 0,33

[0326] Ramachadran Plot

[0327] Statistics**

[0328] Residues 802 602 512

[0329] Most Fevered region 97.18 97.49 96.85

[0330] Allowed Region 2.84 2.34 3,12

[0331] Disallowed Region 0.00 0.17 0.00

[0332] Clashscore 4.86 3.4s 2.53

[0333] GH.24 Loreto 1972 GIL26 Leon 4509 GII.NA1 Loreto 1257

[0334] Data collection

[0335] Space group P120 £121 P12<1

[0336] Ceil dimensions a. bfc (A) 60.75 80.82 70.98 129.44 71.17 78.59 62.70, 71.16, 78.39

[0337] CL £ v (e) 90.00 1 13.82 90.00 9970 103.92 90.00 90.80 110.04 90.00

[0338] Resolution (A) 45.84-174 (177-1.74) 43.69-1.47 (1.507 ,47) 45.38-1.67 (1.69-1.67)

[0339] CCta 0.992 (0.949) 0.995 (0.476) 0.995 (0.453)

[0340] Completeness 98.8 (96.3) 98.8 (93.9) 99.5 (92.5)

[0341] Redundancy 3.0 (3.0) 2.9 (2.8) 4.6 (4.4)

[0342] Refinement

[0343] Resolution (A) 45.13-174 (1.80-174) 48.68-1.47 (1.53-1.4?) 45.38-1.6? (173-1.87)

[0344] No. unique reflections 63597 (622?) 111572 (10859) 75029 (7469)

[0345] RU’ / V 16.0 / 19.0 (19.5 / 24.2) 14.8 / 18.7 (27.4 / 30.9) 18.5 / 21.3 (29.7 / 327)

[0346] No. atoms 5599 549? 5338

[0347] Protein 4896 4796 4808

[0348] Water 763 515 456

[0349] Ligand 95 458 190

[0350] B-factors (A2) 18.79 18.86 2175

[0351] Protein 15.27 16.53 21.02

[0352] Water 25.61 29.38 27.45

[0353] Ligand 34.68 43.77 33.99

[0354] RMS bond length (A) 0.008 0.014 0.011

[0355] RMS bond angle f) 0.96 1.29 1.04

[0356] Ramachadran Plot

[0357] Statistics^

[0358] Residues 617 616 61 B

[0359] Most Favored region 98.90 98.03 97.72

[0360] Atari Region 3.10 1.97 2.28

[0361] Disallowed Region 0.00 0.00 000

[0362] Clashscore 5.6? 3.44 2.28 where / his the mean of / hiobservations of reflection h. Numbers in parenthesis represent highest resolution shell.bR^ and6 *

[0363] 100 far95% of recorded data or S% data (R^). * Determined using MolFrobity

[0364] (10.1002 / pro.333Q) Table 2. Amino add analysis of GII P domain X-ray crystal structures, where the P domain is further subdivided into Pl-1, P2, and Pl-2 subdomains. Table 3. Data collection and refinement statistics for unbound GII.4c P domain and GII.4c

[0365] P domain H2-type complex.

[0366] G!l.4c apo Gll.4c H2 type

[0367] Data collection

[0368] Space group P2^252^ P2i2^

[0369] Ceil dimensions a(% c (A) 75.47, 90.58. SI .33 76.67 . 90.47, 91.34 a, & y R) 90.00, 90.00. 90 90.00, 90.00, 8(100

[0370] Resolution (A) 48.95-1.34 (1.37-1.34) 49,28-1.81 (1.64-1.61)

[0371] 0.072 (1.300) 0.071 (0.726)

[0372] <lto(lj> 10.1 (0.9) 1 1.7 (1 ,7}

[0373] CCW0.999 (0.609) 0.993 (0.769)

[0374] Completeness 99.7 (94.5) 99.9 (98.5)

[0375] Redundancy 8.9 (8.6) 6.8 (6.6)

[0376] Refinement

[0377] Res * it o ' A) 45.67-1.34 (1.39-1.34) 45.67-1.61 (1 .67-1.61 )

[0378] No. j« t, ce reflections 139399 (13446) 82629 (8124)

[0379] OV 13.9 / 16.1 (23.4 / 26.0) 15.7 / 18.5 (21.5 / 26.5)

[0380] No. atoms 5333 5453

[0381] Protein 4720 4729

[0382] Water 512 669

[0383] Ligand 225 98

[0384] B-factors (A2) 24.10 19.59

[0385] Protoin 22.54 18,47

[0386] Water 34.55 26.78

[0387] Ligand 44.28 28 59

[0388] RMS bond length (A) 0.008 0.008

[0389] RMS bond angle (*) 0.98 0.98

[0390] Ramachadran Plot

[0391] Statistics**

[0392] Residues 612 612

[0393] Most Favored region 98 36 98.36

[0394] Aitewed Region 1.64 1.64

[0395] Disallowed Region 0.00 OJOO

[0396] Clashscore 2.23 1 .82 4 is the mean of 4s observations of reflection ft. Numbers in parenthesis represent highest resolution shell.bR?3Cferandc~ F«dl x

[0397] TOO for 95% of recorded data (R^sr) or 5% data (R^). ** Determined using MoJProbity (T0.1002 / pra,3330) References

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[0421] 23. Koromyslova AD, Leuthold MM, Bowler MW, Hansman GS. 2015. The sweet quartet: Binding of fucose to the norovirus capsid. Virology 483 :203-8.10.1016 / j .virol.2015.04.006

[0422] 24. Devant JM, Hansman GS. 2021. Structural heterogeneity of a human norovirus vaccine candidate. Virology 553:23-34.10.1016 / j. virol.2020.10.005

[0423] 25. Salmen W, Hu L, Bok M, Chaimongkol N, Ettayebi K, Sosnovtsev SV, Soni K, Ayyar BV, Shanker S, Neill FH, Sankaran B, Atmar RL, Estes MK, Green KY, Parreno V, Prasad BVV. 2023. A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity. Nat Commun 14:6516.10.1038 / s41467-023- 42146-0 Koromyslova AD, Devant JM, Kilic T, Sabin CD, Malak V, Hansman GS. 2020. Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus. J Virol

[0424] 94.10.1128 / jvi.00660-20 Lindesmith LC, McDaniel JR, Changela A, Verardi R, Kerr SA, Costantini V, Brewer- Jensen PD, Mallory ML, Voss WN, Boutz DR, Blazeck JJ, Ippolito GC, Vinje J, Kwong PD, Georgiou G, Baric RS. 2019. Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus

[0425] V accination . Immunity 50 : 1530- 1541. e 8.10.1016 / j . immuni .2019.05.007 Alvarado G, Salmen W, Ettayebi K, Hu L, Sankaran B, Estes MK, Venkataram Prasad BV, Crowe JE, Jr. 2021. Broadly cross -reactive human antibodies that inhibit genogroup I and II noroviruses. Nat Commun 12:4320.10.1038 / s41467-021-24649-w

[0426] Example 2 - Antigenic structural analysis of bat and human norovirus protruding (P) domains

[0427] Noroviruses are highly contagious, infecting both humans and animals. At least 10 genogroups exist (GI-GX), which are further divided into genotypes with the GII genotypes causing most outbreaks (1). The human norovirus capsid protruding (P) domain binds histo-blood group antigen (HBGA) co-factors for infection (2, 3), and a recently discovered bat norovirus (GX) was also found to interact with HBGAs (4). The genetic variability of the capsid gene is coupled with antigenic diversity and associated with escape from herd immunity, emergence of antigenic variants, and possible changes in host tropism (4-13). In this Example, the P domain structures of four human noroviruses and one bat norovirus were determined using X-ray crystallography to examine structural features linked to potential interspecies transmission.

[0428] The P domains of GII.9 (AY038599), GII.23 (KT290889), GII.27 (MG495077), GVIII (AJ844470), and GX (MF373609) were expressed in E.coli (14). Protein crystals were grown using a sitting drop method (Table 4) and X-ray diffraction was collected as described (15, 16). The five P domains were structurally equivalent with a root mean square deviation for C -alpha atoms ranging between 0.586 A (GII.23 -GII.27) and 1.645 A (GII.9-GX). The principal structural difference was a variable length P2 subdomain loop (Loop A), which was located near the HBGA pocket (Fig. 10A). Two P domain residues that regularly bind HBGAs (e.g., GII.4 R345 and D374) were conserved in GII.9, GII.23, and GII.27 P domains (Fig. 10B). Also, these residue side chains were properly positioned to bind the fucose moiety of HBGAs (structure not shown). These two equivalent residues were lacking in the GVIII and GX P domains; however, an aspartic acid (D367) was located nearby in the GX P domain (Fig. 10B).

[0429] The cross-reactivity of the P domains was determined using direct ELISA with a broadspectrum human norovirus Nanobody (Fc-NB26; SEQ ID NO: 13) that binds to a highly conserved region on the P domain (15, 16). Fc-NB26 bound to GII.9, GII.23, and GII.27 P domains in a dosedependent manner at concentrations less than 80 ng / mL (Fig. 11A). Comparable binding values were observed with other human GII genotypes (GII.l, GII.4, GII.8, GIL 10, GII.14, GII.17, GII.24, GII.26, and GII.NA1) (15, 16). Fc-NB26 bound to the GVIII P domain at weaker concentrations than these GII genotypes, while Fc-NB26 did not bind to the GX P domain at any concentration tested (Fig. 11A). Structural modelling of the Fc-NB26-binding site indicated that GII.9, GII.23, and GII.27 P domains contained most of the equivalent residues that could interact with Fc-NB26 (Fig. 11B and C) (15, 16). The GVIII P domain also had many Fc-NB26-binding residues (Fig. 11D), while most Fc-NB26-binding residues were absent in GX (Fig. HE).

[0430] The present Example demonstrates that Fc-NB26 / NB26 binds most GII genotypes, and the epitope is vulnerable and a therapeutic target region for GII noroviruses (15, 17, 18). Table 4: Data collection and refinement statistics of P domain X-ray crystal structures

[0431] Data collection Space group P 21 21 2 P l 21 1

[0432] Cell dimensions a, b, c (A) 94.296.965.8 60.1 81.071 .8 a, / 3, y D 90.090.090.0 90.0 113.490.0

[0433] Resolution range (A) 48.44- 1.98 (2.03-1 .98) 45.59-1.22 (1 .24-1.22)

[0434] No. unique reflections 42429 (2901 ) 185179 (8742)

[0435] 0.226 (1.766) 0.142 (0.626) 0.237 (1.848) 0.165 (0.717)

[0436] Rpjmc0.070 (0.541) 0.083 (0.344)

[0437] <l / o(l)> 11.75 (1.8) 97 (3.6)

[0438] CC5 / 2 0.996 (0.573) 0.986 (0.774)

[0439] Completeness 99.5 (98.2) 99.2 (94.9)

[0440] Multiplicity 11.3 (11.3) 3.8 (3.8)

[0441] Refinement Resolution range (A) 47.13-1.98 (2.03-1.98) 40.48-1.22 (1.23-1.22)

[0442] Rwos.kw0.1645 (0,2543) 0.1226 (0.1585)

[0443] Rfte8c'* 0.2095 (0.3041) 0.1463 (0.1997)

[0444] No. atoms 5284 11721

[0445] Protein 4725 10203

[0446] Water 557 1345

[0447] Ligand 2 173

[0448] B-factors (A5) 27.37 14.69

[0449] Protein 26.7 10.67

[0450] Water 32.91 29.23

[0451] Ligand 66.74 29.32

[0452] RMS bond length (A) 0.006 0.007

[0453] RMS bond angle (°) 0.83 1

[0454] Ramachandran Plot Statistics^ Residues 605 619

[0455] Most favored region 98.67 97.38

[0456] Allowed region 1.16 2.62

[0457] Disallowed region 0.17 0

[0458] Clashscore 2.27 2.6

[0459] PDB ID 9EDM 9EDN

[0460] Mother solution 0.1 M HEPES pH 7.0, 0.1 M sodium acetate (pH 4.6).

[0461] 30% vol / vol Jeffamine 8% wt / vol PEG 4000

[0462] ED-2003 Table 4 cont’d

[0463] P 1 21 1 P 31 2 1 P 322 1

[0464] 51.290.667.1 107,2 107.261.6 67.567.5 238.8

[0465] 90.0 106.3 90.0 90.090.0 120.0 90.090.0 120.0

[0466] 49.09-1.42 (1.45-1.42) 46.44-1.76 (1.79-1.76) 47.75-1 .63 (1 ,66-1.63)

[0467] 109595 (4942) 40834 (2291) 79623 (12627)

[0468] 0.086 (0.684) 0.104 (0.992) 0.143 (1 ,626)

[0469] 0.094 (0.750) 0.108 (1.030) 0.149 (1.691 )

[0470] 0.039 (0.305) 0.029 (0.273) 0.040 (0.458)

[0471] 12.0 (2.8) 15.4 (1.87) 14.3 (1.8)

[0472] 0.998 (0.870) 0.999 (0.843) 0.999 (0.643)

[0473] 99.5 (90,7) 99.9 (99.0) 99.9 (97.2)

[0474] 5.7 (5.6) 13.8 (13.6) 13.5 (13.4)

[0475] 49.08-1.42 (1.44-1.42) 46.44-1.76 (1.80-1.76) 47.11-1.63 (1 ,65-1.63)

[0476] 0.1316 (0.1919) 0.1395 (0.2201) 0.1624 (0.2421)

[0477] 0.1720 (0.2540) 0.1807 (0,2591) 0.1938 (0.2325)

[0478] 5987 3250 5682

[0479] 5026 2584 4782

[0480] 960 665 880

[0481] 1 1 20

[0482] 25.39 24.13 22.45

[0483] 22.61 20.47 19.96

[0484] 39.93 38.33 35 ^2

[0485] 25.78 28.06 51.15

[0486] 0.005 0.006 0.006

[0487] 0.77 0.82 0.8

[0488] 614 305 594

[0489] 96.7 96.35 98.47

[0490] 3.3 3.32 1 .53

[0491] 0 0.33 0

[0492] 1.22 z.52 2.12

[0493] 9ED0 9EDP 9EDQ

[0494] 0.17 M ammonium sulfate, 0.1 .M Bis-Tns (pH 5.5), 1.6 ,M magnesium sulfate 25.5% wt / vo! PEG 4000 25% wt / vo! PEG 3350 Emerge = Sh Si |Z(h)i - Zh| / Sh Si Z(h)i where Zh is the averaged intensity of all reflections h. bRmeas, = Sh [A7 (TV - 1)] 1 / 2 Si| Z(ih) - Jh | / Sh Si Z(ih). cKpim = Sh [1 / (<V- 1)] 1 / 2 Si | Z(ih) - Zh | / Sh Si Z(ih). iZRwork and eAfree = S|Fobs - Zcalc| / S|Zobs| x 100 for 95% of recorded data (Z?work) or 5% data (Mree).

[0495] / Determined using MolProbity. gX-ray crystallography data were processed using XDS (high-resolution data truncation was guided by the use of the CCI / 2 statistic). Molecular replacement was carried out using Phaser and the structures were refined in Phenix refine. Numbers in parenthesis represent the highest resolution shell.

[0496] References

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[0507] 12. Eden J-S, Tanaka MM, Boni MF, Rawlinson WD, White PA. 2013. Recombination within the pandemic norovirus GII.4 lineage. J Virol 87:6270-6282. https: / / doi.org / 10.1128 / JVI.03464-12

[0508] 13. Lindesmith LC, Beltramello M, Donaldson EF, Corti D, Swanstrom J, Debbink K, Lanzavecchia A, Baric RS. 2012. Immunogenetic mechanisms driving norovirus GII.4 antigenic variation. PLoS Pathog 8:el002705. https: / / doi.org / 10.1371 / journal.ppat.1002705

[0509] 14. Hansman GS, Biertumpfel C, Georgiev I, McLellan JS, Chen L, Zhou T, Katayama K, Kwong PD. 2011. Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability. J Virol 85:6687- 6701. https: / / doi.org / 10.1128 / JVI.00246- 11

[0510] 15. Koromyslova AD, Hansman GS. 2017. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization. PLoS Pathog 13:el006636. https: / / doi.org / 10.1371 / journal.ppat.1006636

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[0512] 17. Koromyslova AD, Hansman GS. 2015. Nanobody binding to a conserved epitope promotes norovirus particle disassembly. J Virol 89:2718-2730. https: / / doi.org / 10.1128 / JVI.03176-14

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[0516] 21. Cao S, Lou Z, Tan M, Chen Y, Liu Y, Zhang Z, Zhang XC, Jiang X, Li X, Rao Z. 2007. Structural basis for the recognition of blood group trisaccharides by norovirus. J Virol 81:5949- 5957. https: / / doi.org / 10.1128 / JVI.00219-07

[0517] 22. Tan M, Hegde RS, Jiang X. 2004. The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors. J Virol 78:6233-6242. https: / / doi.org / 10.1128 / JVI.78.12.6233-6242.2004 23. Huang P, Farkas T, Marionneau S, Zhong W, Ruvoen-Clouet N, Morrow AL, Altaye M, Pickering LK, Newburg DS, LePendu J, Jiang X. 2003. Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: identificationof 4 distinct strain- specificpatterns. J Infect Dis 188:19-31. https: / / doi.org / 10.1086 / 375742 24. Aragao D, Aishima J, Cherukuvada H, Clarken R, Clift M, Cowieson NP, Ericsson DJ, Gee

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Claims

CLAIMS:

1. A polypeptide conjugate comprising:(a) a single domain antibody that is directed against a P domain of a norovirus, wherein the single domain antibody comprises:(i) a CDR1 that comprises the amino acid sequence of RIIFFMYD (SEQ ID NO: 1) or a variant thereof, a CDR2 that comprises the amino acid sequence of QINSDVST (SEQ ID NO: 2) or a variant thereof and a CDR3 that comprises the amino acid sequence of YCNVRRASA (SEQ ID NO: 3) or a variant thereof; or(ii) a CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5), GSIFSIYL (SEQ ID NO: 6) or a variant thereof, a CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and a CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8), KREDFSAYAPPSGS (SEQ ID NO: 9) or a variant thereof; and(b) an Fc region of an immunoglobulin, or a fragment, variant or derivative thereof.

2. The polypeptide conjugate of Claim 1, wherein the single domain antibody comprises the CDR1 that comprises the amino acid sequence of RIIFFMYD (SEQ ID NO: 1) or a variant thereof, the CDR2 that comprises the amino acid sequence of QINSDVST (SEQ ID NO: 2) or a variant thereof and the CDR3 that comprises the amino acid sequence of YCNVRRASA (SEQ ID NO: 3) or a variant thereof.

3. The polypeptide conjugate of Claim 1 or Claim 2, wherein the single domain antibody comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO:

4. or a fragment, variant or derivative thereof.

4. The polypeptide conjugate of Claim 1, wherein the single domain antibody comprises the CDR1 that comprises the amino acid sequence of GSIFSIYA (SEQ ID NO: 5) or a variant thereof, the CDR2 that comprises the amino acid sequence of ISSGGGTN (SEQ ID NO: 7) or a variant thereof and the CDR3 that comprises the amino acid sequence of KREDYSAYAPPSGS (SEQ ID NO: 8) or a variant thereof.

5. The polypeptide conjugate of Claim 4, wherein the single domain antibody comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 10 or 11, or a fragment, variant or derivative thereof.

6. The polypeptide conjugate of any one of the preceding claims, wherein the Fc region comprises, consists of or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 to 34, or a fragment, variant or derivative thereof.

7. The polypeptide conjugate of any one of the preceding claims, wherein the Fc region comprises, consists of or consists essentially of the amino acid sequence set forth in SEQ ID NO: 26, or a fragment, variant or derivative thereof.

8. The polypeptide conjugate of any one of the preceding claims, wherein the single domain antibody is conjugated, connected or otherwise linked to the Fc region by a hinge region.

9. The polypeptide conjugate of Claim 8, wherein the hinge region comprises, consists of or consists essentially of an amino acid sequence set forth in SEQ ID NO: 16, or a fragment, variant or derivative thereof.

10. The polypeptide conjugate of any one of the preceding claims, comprising, consisting of or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 15, or a fragment, variant or derivative thereof.

11. The polypeptide conjugate of any one of the preceding claims, wherein the norovirus is of a GII genogroup.

12. The polypeptide conjugate of any one of the preceding claims, wherein the norovirus is of a GII.l genotype, a GII.4 genotype, a GII.8 genotype, a GII.9 genotype, a GII.10 genotype, a GII.14 genotype, a GII.17 genotype, a GII.23 genotype, a GII.24 genotype, a GII.26 genotype, a GII.27 genotype or a GII.NA1 genotype.

13. The polypeptide conjugate of any one of the preceding claims, wherein the polypeptide conjugate has a KD for the P domain of the norovirus of lower than about 200 nM, lower than about 100 nM, lower than about 70 nM, lower than about 50 nM, lower than about 25 nM, lower than about 10 nM, lower than about 5 nM or lower than about 1 nM.

14. The polypeptide conjugate of any one of the preceding claims, wherein the single domain antibody has been at least partly humanized.

15. The polypeptide conjugate of any one of the preceding claims, further comprising one or more of a detectable marker, a therapeutic agent, a half-life extender and a nanocarrier.

16. An isolated nucleic acid comprising a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, the polypeptide conjugate of any one of Claims 1 to 15.

17. The isolated nucleic acid of Claim 16, comprising, consisting of or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 17 to 20, a fragment, variant or derivative thereof, or a nucleotide sequence complementary thereto.

18. The isolated nucleic acid of Claim 16 or Claim 17, wherein the isolated nucleic acid is or comprises mRNA.

19. A genetic construct comprising: (i) the isolated nucleic acid of any one of Claims 16 to 18; or (ii) a nucleotide sequence complementary thereto; operably linked or connected to one or more regulatory sequences in an expression vector.

20. A host cell transformed with the nucleic acid molecule of any one of Claims 16 to 18 or the genetic construct of Claim 19.

21. A method of producing the polypeptide conjugate of any one of Claims 1 to 15, including the steps of: (i) culturing the previously transformed host cell of Claim 20; and (ii) isolating the polypeptide conjugate from said host cell cultured in step (i).

22. A composition comprising the polypeptide conjugate of any one of Claims 1 to 15, the isolated nucleic acid of any one of Claims 16 to 18, the genetic construct of Claim 19 or the host cell of Claim 20 and optionally a pharmaceutically acceptable carrier, diluent or excipient.

23. A method of diagnosing or monitoring a norovirus infection and / or a disease, disorder or condition associated therewith in a subject, said method including the step of contacting the subject and / or a biological sample from the subject with the polypeptide conjugate of any one of Claims 1 to 15 or the composition of Claim 22.

24. The method of claim 23, which further includes the step of detecting and / or measuring a level of antigen binding to the polypeptide conjugate.

25. A method of inhibiting or preventing binding of a norovirus to a histo-blood group antigen (HBGA) and / or a bile acid in a subject, said method including the step of administering to the subject a therapeutically effective amount of the polypeptide conjugate of any one of Claims 1 to 15, the isolated nucleic acid of any one of Claims 16 to 18, the genetic construct of Claim 19, the host cell of Claim 20 or the composition of Claim 22 to thereby inhibit or prevent binding of the norovirus to the HBGA and / or the bile acid in the subject.

26. A method of inactivating or neutralizing a norovirus in a subject, said method including the step of administering to the subject a therapeutically effective amount of the polypeptide conjugate of any one of Claims 1 to 15, the isolated nucleic acid of any one of Claims 16 to 18, the genetic construct of Claim 19, the host cell of Claim 20 or the composition of Claim 22 to thereby inactivate or neutralize the norovirus in the subject.

27. A method of treating, ameliorating or preventing a norovirus infection and / or a disease, disorder or condition associated therewith in a subject including the step of administering a therapeutically effective amount of the polypeptide conjugate of any one of Claims 1 to 15, the isolated nucleic acid of any one of Claims 16 to 18, the genetic construct of Claim 19, the host cell of Claim 20 or the composition of Claim 22 to thereby treat or prevent the norovirus infection and / or disease, disorder or condition associated therewith in the subject.

28. Use of the polypeptide conjugate of any one of Claims 1 to 15, the isolated nucleic acid of any one of Claims 16 to 18, the genetic construct of Claim 19, the host cell of Claim 20 or the composition of Claim 22 for therapy.

29. Use of the polypeptide conjugate of any one of Claims 1 to 15, the isolated nucleic acid of any one of Claims 16 to 18, the genetic construct of Claim 19, the host cell of Claim 20 or the composition of Claim 22 in the manufacture of a medicament for the treatment and / or prevention of a norovirus infection and / or a disease, disorder or condition associated therewith in a subject.

30. A method for inactivating or neutralising a norovirus associated with a substrate or surface, said method including the step of contacting the substrate with an effective amount of thepolypeptide conjugate of any one of Claims 1 to 15, or the composition of Claim 22 to thereby inactivate or neutralise the norovirus associated with the substrate or surface.

31. A kit or device for diagnosing or monitoring a norovirus infection, and / or a disease, disorder or condition associated therewith, in a subject, said kit or device including the polypeptide conjugate of any one of Claims 1 to 15 or the composition of Claim 22; optionally one or more reagents for detecting the polypeptide conjugate; and optionally instructions for use.

Images