Anti-human norovirus antibody

Abstract

To provide an anti-norovirus antibody that exhibits specific binding activity to norovirus particles of genogroups GI and GII.SOLUTION: An antibody binding to noroviruses of genogroups GI and GII, the antibody having one or more structural domains comprising CDRs represented by the following (a) or (b): (a) a CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO:1); a CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO:2); and a CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO:3); (b) a CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO:7); a CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO:8); and a CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO:9).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anti-human norovirus antibodies that exhibit specific binding activity to GI and GII type norovirus particles. [Background technology]

[0002] Noroviruses are non-enveloped, spherical viruses with a diameter of approximately 30 to 40 nm that belong to the Caliciviridae family. Noroviruses cause acute gastroenteritis symptoms, such as vomiting and diarrhea, in humans. Human noroviruses can cause outbreaks when introduced into congregate facilities such as schools, medical facilities, and food-handling establishments, and can cause food poisoning if they contaminate food. The norovirus genome is a single-stranded (+) RNA of approximately 7.6 kb. Based on genome homology, noroviruses have recently been classified into seven genogroups: genogroup 1 (GI), genogroup 2 (GII), genogroup 3 (GIII), genogroup 4 (GIV), genogroup 5 (GV), genogroup 6 (GVI), and genogroup 7 (GVII). Of these, GI is infectious to humans; GII is infectious to humans and pigs; GIII is infectious to cattle; GIV is infectious to humans, cats, and dogs; GV is infectious to mice; and GVI and GVII are infectious to dogs (see Non-Patent Document 1).

[0003] There are multiple genotypes within each gene group of norovirus, and for noroviruses that are infectious to humans (hereinafter referred to as human noroviruses), genotypes GI.1 to GI.9 have been reported as GI noroviruses, and at least genotypes GII.1 to GII.10, GII.12 to GII.17, and GII.20 to GII.22 have been reported as GII noroviruses. The norovirus genome mutates very rapidly, and it has been reported that recombination occurs between viruses of different genotypes, and the emergence of noroviruses with new genotypes has also been confirmed.

[0004] Antibodies are used to detect viral proteins, such as antigen testing methods that use immunochromatography or ELISA (Enzyme-linked immunosorbent assay) as their measurement principles. In the case of norovirus, each genotype exhibits different antigenicity, but the same genotype is said to exhibit the same antigenicity. Furthermore, the VP1 protein, a structural protein of norovirus, has regions where the amino acid sequence is conserved for each gene group. For this reason, antigen testing kits for norovirus that use immunochromatography or ELISA as their measurement principles are also commercially available.

[0005] On the other hand, most commercially available antigen test kits use monoclonal or polyclonal IgG antibodies derived from mice, rabbits, etc. VHH (variable domain of heavy chain of heavy chain antibody) is a single-domain antibody consisting only of the variable domain of the heavy chain antibody of camelids. While VHH exhibits binding activity equivalent to that of IgG antibodies, it has the following characteristics: (1) its molecular weight is approximately one-tenth that of IgG antibodies, and it is expected to bind to novel epitopes that conventional antibodies cannot bind; (2) unlike IgG antibodies, it has a highly reversible protein structure and is highly resistant to heat and pressure; and (3) unlike IgG antibodies, it can be produced using microorganisms such as yeast and bacteria. Furthermore, (4) VHH is highly compatible with in vitro antibody selection techniques such as cDNA display and phage display, and can be developed in a shorter time than IgG antibodies obtained by immunization of mice, rabbits, etc. Therefore, VHHs are expected to be used as alternative antibodies to conventional immunoglobulins such as IgG and IgA antibodies, which have been used in pharmaceuticals and diagnostic reagents.

[0006] VHHs also have clear advantages in the development of antigen test kits. In particular, the following properties of VHHs are considered to be advantageous compared to IgG antibodies: (1) VHHs can be densely immobilized on particles or nitrocellulose membranes, allowing for the presentation of more paratopes on the substrate; (2) VHHs have excellent protein stability, which allows for the expected longer shelf life of products; (3) VHHs can be mass-produced inexpensively using microorganisms, thereby further reducing manufacturing costs; and (4) VHHs lack the Fc region, which can cause nonspecific reactions in immunochromatography.

[0007] As such, VHHs have many advantages in terms of properties and production compared to conventional antibodies (such as IgG antibodies), and therefore detection technologies using single-domain antibodies targeting norovirus can be offered with advantages in terms of performance and cost compared to conventional technologies using IgG antibodies.

[0008] For antigen test kits, it is important to obtain monoclonal antibodies that react with a wider range of human norovirus gene groups and genotypes. As a single domain antibody, Nano-85 has been reported to react with GII.1, GII.2, GII.4, GII.12, and GII.17 noroviruses (Non-Patent Document 2).

[0009] However, since nine genotypes of GI norovirus and at least 19 genotypes of GII norovirus have been reported for human norovirus, there is a need to develop VHHs that react with a greater number of genotypes of human norovirus. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Advances in laboratory methods for detection and typing of norovirus. J Clin Microbiol. 2015;53(2):373-81. [Non-patent document 2] Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization. PLoS Pathog. 2017;13(11):e1006636. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention relates to providing anti-norovirus antibodies that exhibit specific binding activity to GI and GII type norovirus particles. [Means for solving the problem]

[0012] The inventors conducted studies to obtain single-domain antibodies that exhibit binding activity to virus-like particles (VLPs) of GII.4 or GII.1 noroviruses. As a result, they succeeded in obtaining clones that bind to multiple genotypes of noroviruses belonging to GI and GII types by screening a VHH antibody library containing CDRs 1 to 3 with specific amino acid numbers in the construct using cDNA display methods.

[0013] That is, the present invention relates to the following 1) to 7). 1) An antibody that binds to GI and GII noroviruses and has one or more structural domains containing the CDRs shown in (a) or (b) below: (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). 2) A nucleic acid encoding the antibody of 1). 3) A mixture of antibodies that bind to GI and GII noroviruses, comprising one or more antibodies selected from those that bind to noroviruses and have one or more structural domains containing CDRs as shown in (a) and (b) below, and an antibody that binds to noroviruses and has one or more structural domains containing CDRs as shown in (c) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by GFSVNSLTMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GMNKDGSTS (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by QGPLFRPDLPLYDS (SEQ ID NO: 6) 4) A method for detecting human norovirus in a sample, comprising the step of contacting a test sample with an antibody that binds to GI and GII noroviruses and has one or more structural domains containing CDRs shown in (a) or (b) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). 5) A method for detecting human norovirus in a sample, comprising the step of contacting a test sample with a mixture of antibodies that bind to GI and GII noroviruses, the mixture including one or more antibodies selected from those that bind to noroviruses having one or more structural domains containing CDRs as shown in (a) and (b) below, and antibodies that bind to noroviruses having one or more structural domains containing CDRs as shown in (c) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by GFSVNSLTMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GMNKDGSTS (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by QGPLFRPDLPLYDS (SEQ ID NO: 6) 6) A human norovirus detection kit containing the antibody of 1) or the antibody mixture of 3). 7) A preventive or therapeutic drug for human norovirus infection containing the antibody of 1) or the antibody mixture of 3). [Effects of the Invention]

[0014] The present invention provides single-domain antibodies and antibody mixtures that are highly reactive with GI and GII norovirus particles. Because the antibodies and antibody mixtures of the present invention exhibit excellent specificity for norovirus particles of multiple genotypes belonging to GI and GII types, the use of these antibodies and antibody mixtures enables the specific detection of norovirus, i.e., the rapid detection of norovirus infection. Furthermore, by testing a norovirus-positive sample for norovirus using these antibodies and antibody mixtures and confirming no reaction, it is possible to confirm that the norovirus contained in the sample is human norovirus. DETAILED DESCRIPTION OF THE INVENTION

[0015] The norovirus-binding antibody of the present invention (hereinafter referred to as "the antibody of the present invention") is an anti-norovirus monoclonal antibody having one or more structural domains including CDR1 to 3 shown in (a) to (c) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by GFSVNSLTMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GMNKDGSTS (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by QGPLFRPDLPLYDS (SEQ ID NO: 6)

[0016] Norovirus is a non-enveloped, spherical virus with a diameter of approximately 30 to 40 nm that belongs to the Caliciviridae family and causes norovirus infection. The antibody of the present invention is an antibody that binds to human norovirus, and more specifically, an antibody that binds to the structural proteins of the norovirus and complexes thereof. These complexes include virus-like particles (VLPs), VP1 protein, and complexes thereof.

[0017] The structural domain of the antibody of the present invention has three CDRs: CDR1, CDR2, and CDR3. A CDR (Complementarity Determining Region) contains a sequence-variable antigen recognition site or a random sequence region, and is also called a hypervariable region. In the structural domain of the antibody of the present invention, the three CDRs are present in the order of CDR1, CDR2, and CDR3 from the N-terminus.

[0018] The binding ability to norovirus can be evaluated by methods known to those skilled in the art. Specifically, as shown in the Examples below, it can be evaluated by determining the equilibrium dissociation constant KD using surface plasmon resonance. It can also be evaluated by methods such as ELISA, immunochromatography, isothermal titration calorimetry, and biolayer interferometry.

[0019] The structural domain of the antibody of the present invention may have framework regions at both ends of CDR1, CDR2, and CDR3. The framework region is a highly conserved region in the variable region of an antibody molecule, excluding the complementarity-determining regions. That is, in one embodiment, the structural domain of the present invention has, in this order, a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework region (FR4). Examples of the amino acid sequences of the framework regions in the structural domains include the amino acid sequences shown below or amino acid sequences having 80% or more identity to the amino acid sequences. FR1: AEVQLVESGGGLVQPGGSLRLSCVPS (SEQ ID NO: 10) AEVQLVESGGGLVQPGGSQRLSCTAS (SEQ ID NO: 11) AEVQLVESGGGLVQPGGSLRLSCATS (SEQ ID NO: 12) FR2: WFRQAPGKEREFVA (SEQ ID NO: 13) WVRLPQGKQLEYVG (SEQ ID NO: 14) WVRQTPEKGLEWVA (SEQ ID NO: 15) FR3: YADSVKGRFTISRDNDKSTVYLQMNSLRPEDSGIYYCRS (SEQ ID NO: 16), YMDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCNA (SEQ ID NO: 17), YAEAVKGRFTISRDNVKNTLYLQMNSLKPEDTAVYYCNA (SEQ ID NO: 18) FR4: WGQGTQVTVSS (SEQ ID NO: 19)

[0020] Examples of framework regions consisting of amino acid sequences having 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 10 to 19 include framework regions consisting of amino acid sequences having preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more identity.

[0021] Here, the identity of amino acid sequences refers to the percentage (%) of the number of positions where identical amino acid residues exist in two amino acid sequences when the two sequences are aligned, relative to the total number of amino acid residues in both sequences. The identity of sequences can be calculated, for example, by performing an analysis using BLAST (Basic Local Alignment Search Tool) from the National Center for Biotechnology Information (NCBI).

[0022] Among the antibodies of the present invention, an antibody having a structural domain (SEQ ID NO: 20) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, wherein CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, CDR3 is the amino acid sequence shown in SEQ ID NO: 3, FR1 is the amino acid sequence shown in SEQ ID NO: 10, FR2 is the amino acid sequence shown in SEQ ID NO: 13, FR3 is the amino acid sequence shown in SEQ ID NO: 16, and FR4 is the amino acid sequence shown in SEQ ID NO: 19; an antibody having a structural domain (SEQ ID NO: 22) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 7, CDR2 is the amino acid sequence shown in SEQ ID NO: 8, CDR3 is the amino acid sequence shown in SEQ ID NO: 9, FR1 is the amino acid sequence shown in SEQ ID NO: 12, FR2 is the amino acid sequence shown in SEQ ID NO: 15, FR3 is the amino acid sequence shown in SEQ ID NO: 18, and FR4 is the amino acid sequence shown in SEQ ID NO: 19; Antibodies having a structural domain (SEQ ID NO: 21) in which CDR1 is the amino acid sequence shown in SEQ ID NO: 4, CDR2 is the amino acid sequence shown in SEQ ID NO: 5, CDR3 is the amino acid sequence shown in SEQ ID NO: 6, and FR1 is the amino acid sequence shown in SEQ ID NO: 11, FR2 is the amino acid sequence shown in SEQ ID NO: 14, FR3 is the amino acid sequence shown in SEQ ID NO: 17, and FR4 is the amino acid sequence shown in SEQ ID NO: 19 are clones highly reactive to norovirus structural proteins (SEQ ID NO: 20 is NoVHH204, SEQ ID NO: 22 is NoVHH26, and SEQ ID NO: 21 is NoVHH205) obtained by screening using the cDNA display method in the examples described below, and are suitable anti-norovirus monoclonal antibodies.

[0023] NoVHH204 binds to GI types (1, 2, 6, 7, 8, 9) and GII types (1, 4, 9, 15, 21) of noroviruses, NoVHH26 binds to GI types (2, 4, 6, 7, 8, 9) and GII types (1, 2, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 21, 22) of noroviruses, and NoVHH205 binds to GII types (3, 4, 10, 22) of noroviruses.

[0024] The form of the antibody of the present invention is not limited as long as it has at least one of the above-mentioned structural domains, and it can be a single-domain antibody (also called a nanobody) or a multimer (e.g., a dimer) in which multiple single-domain antibodies are linked. Multimers include multimers in which multiple structural domains of the present invention are linked, as well as multimers in which one or more of the structural domains are linked to one or more other structural domains that have antigen specificities different from those of the structural domains. A single-domain antibody refers to an antibody that has the property of specifically binding to an antigen via a single variable region (antigen-binding domain). Single-domain antibodies include antibodies whose variable region consists only of the variable region of a heavy chain (heavy-chain single-domain antibodies) and antibodies whose variable region consists only of the variable region of a light chain (light-chain single-domain antibodies). VHH, a heavy-chain antibody identified in camelids (e.g., camel, llama, alpaca, etc.), and VNAR, a heavy-chain antibody derived from cartilaginous fish (e.g., shark), are known as types of single-domain antibodies, and VHH is preferred in the present invention. Furthermore, the antibodies of the present invention may be modified to evade the human immune system. Antibodies modified to evade the human immune system can be administered to humans and therefore can be used as pharmaceuticals.

[0025] The method for producing the antibodies of the present invention is not particularly limited and they can be easily produced by techniques known in the art. For example, they can be produced by combining solid-phase peptide synthesis with native chemical ligation (NCL) or by genetic engineering. However, a preferred method involves designing an artificial gene optimized for expression of the antibody of interest in host cells by subjecting nucleic acids encoding the antibodies of the present invention to processes such as codon optimization, incorporating the artificial gene into an appropriate vector, and introducing the vector into host cells to produce the recombinant antibody.

[0026] Host cells used in producing recombinant antibodies include, for example, Escherichia coli, Bacillus subtilis, fungi, animal cells, plant cells, baculovirus / insect cells, and yeast cells. Expression vectors suitable for various host cells can be used to express antibodies. Examples of expression vectors that can be used include E. coli-derived vectors such as pBR322, pBR325, pUC12, and pUC13; Bacillus subtilis-derived vectors such as pUB110, pTP5, and pC194; shuttle vectors compatible with both E. coli and Bacillus subtilis, such as pHY300PLK; yeast-derived vectors such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and baculovirus; and vectors modified from these. These expression vectors have a replication origin, a selection marker, and a promoter appropriate for each vector, and may also have, as necessary, an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, etc. Furthermore, to facilitate purification of the expressed polypeptide, the expression vector may have inserted therein a nucleotide sequence for expressing a fused tag such as a FLAG tag, His tag, HA tag, or GST tag.

[0027] When the expressed antibody of the present invention is extracted from cultured bacterial cells or cells, the bacterial cells or cultured cells are collected after cultivation by a known method, suspended in an appropriate buffer, and disrupted by ultrasound, lysozyme, and / or freeze-thawing, followed by centrifugation or filtration to obtain a soluble extract. The desired antibody can be obtained from the obtained extract by an appropriate combination of known separation and purification methods. Known separation and purification methods include methods that utilize solubility, such as salting out and solvent precipitation; methods that primarily utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-PAGE; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize specific affinity, such as affinity chromatography; methods that utilize differences in hydrophobicity, such as reversed-phase high-performance liquid chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing.

[0028] The antibody (a) of the present invention binds to six GI type and five GII type norovirus structural proteins, the antibody (b) binds to six GI type and 14 GII type norovirus structural proteins, and the antibody (c) binds to four GII type norovirus structural proteins. Therefore, by contacting an antibody (a) or (b) of the present invention, or an antibody mixture containing one or more antibodies selected from the antibodies (a) and (b) and the antibody (c), with a test sample that contains or may contain norovirus, it is possible to confirm with high certainty the presence or absence of GI type and GII type norovirus in the sample. Specifically, detection of norovirus using the antibody of the present invention comprises the steps of contacting the antibody of the present invention with a test sample to form a complex between the antibody of the present invention and the norovirus in the test sample, and detecting the norovirus in the complex. Furthermore, the antibody of the present invention can also be used to detect virus-specific antibodies in serum by adding a portion of an antigen containing a structural protein of norovirus to anti-norovirus antibodies (e.g., serum antibodies) contained in an immobilized test sample (serum) to allow binding, and confirming the presence of norovirus antigens in this bound state.

[0029] Test samples include, but are not limited to, biological samples (e.g., saliva, body fluids, blood, serum, urine, feces, tissues, cells, tissue or cell fragments, etc.), as well as solutions and solid surfaces that may be contaminated with norovirus. In terms of binding to VLPs, it is preferable to dissolve the virus in the test sample in a solution containing a surfactant or the like and allow it to bind to the antibody in the solution. The antibody may or may not be immobilized on a solid phase. The step of detecting norovirus in the conjugate can be carried out, for example, by reacting the conjugate with an anti-norovirus antibody that recognizes an epitope different from that of the antibody of the present invention in the conjugate. Alternatively, norovirus in the conjugate can be detected in a liquid phase by a homogeneous assay.

[0030] Furthermore, the antibodies of the present invention can be used as components of kits for detecting human norovirus, which can be used as diagnostic agents for infections caused by human norovirus, specifically GI and GII types of norovirus, and as tools for developing preventive or therapeutic agents for infections caused by human norovirus, specifically GI and GII types of norovirus. The detection kit may include, in addition to the anti-norovirus antibody of the present invention, reagents and instruments necessary for detection, such as an antibody that recognizes the antibody of the present invention or an anti-norovirus antibody that recognizes an epitope different from the antibody of the present invention, a solid phase support, a buffer solution, an enzyme reaction stop solution, a microplate reader, etc. In the detection kit, the antibody of the present invention may be immobilized on a solid phase, such as beads, a membrane, the side or bottom surface of a reaction vessel, a plate-like substrate such as a slide glass, or a well substrate such as an immunoplate, to which the antibody of the present invention is directly or indirectly immobilized.

[0031] In such a preventive or therapeutic agent for human norovirus infection, the content of the antibody of the present invention in the composition can be adjusted appropriately. Furthermore, such a pharmaceutical can be applied by administering an effective amount of the antibody of the present invention to a patient once to several times a week, preferably by intravenous injection, infusion, etc.

[0032] In the present invention, the following aspects are further disclosed in relation to the above-described embodiment. <1> An antibody that binds to GI and GII noroviruses, which has one or more structural domains containing the CDRs shown in (a) or (b) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). <2> binds to a norovirus structural protein, preferably a viral structural protein consisting of norovirus VP1; <1> antibodies. <3> a single domain antibody or a multimer thereof; <1> or <2> antibodies. <4> the single domain antibody is a VHH antibody; <3> antibodies. <5> A multimer of a single domain antibody is a multimer, preferably a dimer, in which multiple structural domains are linked together. <3> antibodies. <6> A single domain antibody multimer is a multimer in which one or more of the structural domains are linked to one or more structural domains having antigen specificity different from that of the structural domains. <3> antibodies.

[0033] <7> The structural domain has, in this order, a first framework region (FR1), CDR1, a second framework region (FR2), CDR2, a third framework region (FR3), CDR3, and a fourth framework region (FR4). <1> ~ <6> Any of the antibodies. <8> FR1 to FR4 consist of the following amino acid sequences: <7> antibodies. FR1: the amino acid sequence shown in SEQ ID NO: 10 or 12 or an amino acid sequence having 80% or more identity with said amino acid sequence FR2: the amino acid sequence shown in SEQ ID NO: 13 or 15 or an amino acid sequence having 80% or more identity with said amino acid sequence FR3: the amino acid sequence shown in SEQ ID NO: 16 or 18 or an amino acid sequence having 80% or more identity with said amino acid sequence FR4: the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 80% or more identity to said amino acid sequence <9> a structural domain (SEQ ID NO: 20) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 1, CDR2 is the amino acid sequence shown in SEQ ID NO: 2, and CDR3 is the amino acid sequence shown in SEQ ID NO: 3, FR1 is the amino acid sequence shown in SEQ ID NO: 10, FR2 is the amino acid sequence shown in SEQ ID NO: 13, FR3 is the amino acid sequence shown in SEQ ID NO: 16, and FR4 is the amino acid sequence shown in SEQ ID NO: 19; <7> antibodies. <10> a structural domain (SEQ ID NO: 22) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 is the amino acid sequence shown in SEQ ID NO: 7, CDR2 is the amino acid sequence shown in SEQ ID NO: 8, and CDR3 is the amino acid sequence shown in SEQ ID NO: 9, FR1 is the amino acid sequence shown in SEQ ID NO: 12, FR2 is the amino acid sequence shown in SEQ ID NO: 15, FR3 is the amino acid sequence shown in SEQ ID NO: 18, and FR4 is the amino acid sequence shown in SEQ ID NO: 19; <7> antibodies. <11> <1> ~ <10> A nucleic acid encoding any one of the antibodies. <12> An antibody mixture that binds to GI and GII noroviruses, comprising one or more antibodies selected from those that bind to noroviruses and have one or more structural domains containing CDRs as shown in (a) and (b) below, and an antibody that binds to noroviruses and has one or more structural domains containing CDRs as shown in (c) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by GFSVNSLTMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GMNKDGSTS (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by QGPLFRPDLPLYDS (SEQ ID NO: 6) <13> The antibody shown in (c) has a structural domain (SEQ ID NO: 21) in which FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are linked in this order, CDR1 having the amino acid sequence shown in SEQ ID NO: 4, CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and CDR3 having the amino acid sequence shown in SEQ ID NO: 6, FR1 having the amino acid sequence shown in SEQ ID NO: 11, FR2 having the amino acid sequence shown in SEQ ID NO: 14, FR3 having the amino acid sequence shown in SEQ ID NO: 17, and FR4 having the amino acid sequence shown in SEQ ID NO: 19. <12> antibody mixture. <14> A method for detecting norovirus in a sample, comprising the step of contacting a test sample with an antibody that binds to GI and GII noroviruses and has one or more structural domains containing CDRs shown in (a) or (b) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). <15> A method for detecting human norovirus in a sample, comprising the step of contacting a test sample with a mixture of antibodies that bind to GI and GII type noroviruses, the mixture including one or more antibodies selected from those that bind to noroviruses having one or more structural domains containing CDRs as shown in (a) and (b) below, and antibodies that bind to noroviruses having one or more structural domains containing CDRs as shown in (c) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3) (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by GFSVNSLTMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GMNKDGSTS (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by QGPLFRPDLPLYDS (SEQ ID NO: 6) <16> <1> ~ <10> or <12> or <13> A norovirus detection kit containing an antibody mixture of the above. <17> <1> ~ <10> or <12> or <13> A preventive or therapeutic drug for norovirus infection containing an antibody mixture of the above. [Example]

[0034] (Example 1) Preparation of virus-like hollow particles of norovirus 1-1. Cell lines, media, and primers used The bacterial strains and cell lines used in this study are listed in Table 1. DH5α and DH10bac were cultured in LB medium (polypeptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7-7.4, Wako Pure Chemical Industries). Agar medium contained 15 g / L agarose (Wako Pure Chemical Industries). Culture was performed at 37°C, with shaking (200 rpm) as needed. Antibiotics were added as needed during transformation. Sf9 was cultured in Sf-900III SFM (Thermo Fisher Scientific). 10 mL of 10,000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) was added per liter of medium (hereafter referred to as Sf900 medium). H5 was cultured in Express Five SFM (Thermo Fisher Scientific). 10 mL of 10,000 U / mL Pencillin-streptomycin (Thermo Fisher Scientific) and 100 mL of 200 mM L-Glutamine (Thermo Fisher Scientific) were added per liter of medium (hereafter referred to as H5 medium). Both Sf9 and H5 were cultured at 27°C. Subculture of the cells was performed in a static culture system using T flasks (Becton Dickinson). Baculovirus amplification and VLP production were carried out in a suspension culture system using Erlenmeyer flasks.

[0035] [Table 1]

[0036] 1-2. Artificial synthesis of genes The sequences of ORF2 and ORF3, encoding norovirus capsid proteins, were obtained from the public gene databases NoroNet (https: / / www.rivm.nl / en / noronet) and NCBI (https: / / www.ncbi.nlm.nih.gov / ). The strain names, accession numbers, and other information for the obtained virus genotypes are listed in Table 2. For genotypes for which sequences were available, artificial sequences were synthesized by inserting four bases upstream of the ORF2 start codon (GI type: GTAA, GII type: GTGA) at the 5' end of the ORF2 / ORF3 sequence and the 3'-untranslated region (UTR, Table 3) of each genotype at the 3' end. For GII.17, an artificial sequence was synthesized by inserting the attL1 sequence (SEQ ID NO: 40) four bases upstream of the ORF2 start codon and a 30-base polyadenine sequence 55 bases downstream of the ORF3 stop codon, followed by the attL2 sequence (SEQ ID NO: 42). For GII.2, GII.3, and GII.17, we commissioned Genscript to synthesize the pUC57 vector with the desired construct inserted. For other genotypes, we commissioned FASMAC to synthesize the pUCFa vector with the desired construct inserted. For constructs other than GI.1, GII.2, GII.3, GII.4, and GII.17, we optimized the sequences to match the codon frequency of the H5 cells used for protein expression.

[0037] [Table 2]

[0038] [Table 3]

[0039] 1-3. Insertion of NoV gene into pDEST8 vector Using a plasmid vector containing the artificially synthesized construct as a template, each NoV gene fragment was amplified by PCR using the primers listed in Table 4. Additionally, a linearized pDEST8 vector was obtained using the pDEST8 vector (Thermo Fisher Scientific) as a template and inverse PCR primers (SEQ ID NO: 68, SEQ ID NO: 69). KOD Plus Neo (TOYOBO) was used as the PCR enzyme. Each NoV gene fragment was inserted into the pDEST8 vector using the In-Fusion® HD Cloning Kit (Takara Bio Inc.). The NoV gene fragment, linearized pDEST8 vector, and reaction reagents listed in Table 5 were mixed and incubated at 50°C for 15 minutes to obtain an In-Fusion reaction solution. For GII.17, equal amounts of the pUC57 vector and pDEST8 vector containing the artificially synthesized gene were mixed as listed in Table 6 and then incubated at 25°C for 1 hour. After the reaction, 1 μL of Proteinase K (manufactured by Takara Bio Inc.) was added and the mixture was reacted at 37° C. for 10 minutes to obtain an LR reaction product.

[0040] [Table 4]

[0041] [Table 5]

[0042] [Table 6]

[0043] 1-4. Transformation of DH5α 2.5 μL of the infusion reaction mixture was added to 100 μL of DH5α competent cells thawed on ice and mixed gently. 5 μL was added for the LR reaction product. After 5 minutes on ice, the cells were incubated at 42°C for 45 seconds and then left on ice for 2 minutes. 1 mL of LB medium was added, and the appropriately diluted solution was plated on LB agar medium containing 100 μg / mL ampicillin. After overnight incubation at 37°C, the resulting colonies were picked and cultured overnight in LB medium containing 100 μg / mL ampicillin. Plasmids were extracted from the culture medium using a QIAprep Spin Miniprep Kit (QIAGEN), and the pDEST8 vector containing the desired construct was obtained.

[0044] 1-5. Construction of recombinant Bacmid (rBacmid) DH10Bac competent cells were thawed on ice, and 1 μL of pDEST8 vector solution (final concentration: 100 ng) was added to 50 μL of competent cells. The pDEST8 vector was introduced by heat shock as in 1-4. After heat shock, 900 μL of SOC medium was added to the cells, and recovery culture was performed at 37°C and 200 rpm for 4 hours. Appropriately diluted bacterial solution was plated on LB agar medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, 10 μg / mL tetracycline, 40 μg / mL IPTG, and 100 μg / mL X-gal. After static culture at 37°C, white colonies were picked on LB agar medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline. Transformants containing the target sequence were selected by PCR. The transformant of interest was cultured in LB medium containing 50 μg / mL kanamycin, 7 μg / mL gentamicin, and 10 μg / mL tetracycline, and then rBacmid was extracted from the culture medium using a QIAprep Spin Miniprep Kit.

[0045] 1-6. Introduction of rBacmid into insect cells To obtain recombinant baculovirus (rBV) carrying the target construct, rBacmid was transfected into Sf9 cells. 8.5 x 10 cells were placed in a 6-well plate (Becton Dickinson). 6 Sf9 cells were seeded at 100 cells / well and allowed to settle on the bottom of the plate. While the cells were settling, the transfection reagent was prepared as follows: 8 μL of Cellfectin Reagent (Thermo Fisher Scientific) was mixed with 100 μL of Grace's insect medium, unsupplemented (Thermo Fisher Scientific). 100 μL of rBacmid solution, prepared at 25 μg / mL in Grace's insect medium, unsupplemented, was added to this solution, mixed gently by pipetting, and then incubated at room temperature for 15 minutes. Cell settling was confirmed under a microscope, and the medium was removed. Plating medium (1.5 mL Grace's insect medium supplemented with 10% FBS, 8.5 mL Grace's insect medium unsupplemented (Thermo Fisher Scientific)) was added at 2.5 mL per well. The prepared transfection reagent was added to the entire well and incubated at 27°C for 3 to 5 hours. The supernatant was removed, and 2 mL of Sf900 medium was added. To prevent evaporation of the culture medium, the wells were wrapped in plastic wrap and protected from light with aluminum foil. The wells were then cultured statically at 27°C for 1 week. The collected culture medium was centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was used as P0 rBV seed.

[0046] 1-7. Amplification of rBV seeds 4 x 10 ml of 250 mL Erlenmeyer flask (Nalgene) 530 mL of Sf9 cell suspension prepared at 1000 cells / mL was added. 1 mL of P0 rBV seed solution was added and cultured at 27°C and 125 rpm for 1 week with shaking. The culture was centrifuged at 8000 rpm for 10 min at 4°C, and the collected supernatant was used as P1 rBV seed. The infectious titer of the rBV seed was measured by plaque assay. The obtained rBV seed was dispensed into 1 mL aliquots and frozen and stored at -80°C. If the infectious titer of the rBV seed was insufficient, further amplification was performed using the same procedure as above, if necessary.

[0047] 1-8. Plaque assay-1 2.4x10 cells in a 6-well plate 6 Sf9 cells were seeded at 100 cells / well. After allowing the cells to settle at room temperature, the supernatant was removed and 900 μL of fresh Sf900 medium was added. 100 μL of rBV seed solution diluted 1:10 with Sf900 medium was added to each well. The plates were shielded from light with aluminum foil and incubated at 27°C for 1 hour with gentle agitation every 15 minutes. The supernatant was removed, and 3 mL of Overlay 1 medium, prepared as described in Table 7, was added to each well. After allowing the plate to stand at room temperature until solidified, the plate was wrapped in plastic wrap and aluminum foil, inverted, and incubated at 27°C for 4 days.

[0048] [Table 7]

[0049] 1-9. Plaque Assay-2 Reagents were mixed as shown in Table 8 to prepare Overlay 2 medium. 2 mL of Overlay 2 medium was layered onto each well and allowed to solidify at room temperature. The plate was wrapped in plastic wrap and aluminum foil, inverted, and incubated at 27°C for 2 days. Once plaques were visible, white light was shone onto the bottom of the plate, and the plaques in the wells with the appropriate dilution ratio were counted. The average plaque counts from two wells were multiplied by the dilution ratio, and then multiplied by 10 to obtain the infectious titer (pfu / mL) of the rBV seed.

[0050] [Table 8]

[0051] 1-10.VLP production VLP production was carried out in a suspension culture system using a 250 mL Erlenmeyer flask. 6 The H5 cell suspension prepared in H5 medium to give a concentration of 100 cells / mL and rBV seeds were added to a 250 mL Erlenmeyer flask, and VLP production was carried out in a 40 mL culture volume. The rBV seeds were added to the culture medium so that the MOI (multiplicity of infection) was 0.01 or higher. The flask was shielded from light with aluminum foil, and the cells were cultured with shaking at 27°C and 125 rpm for one week. Two days after the start of culture, protease inhibitor (Roche) was added. One tablet of protease inhibitor was dissolved in 1 mL of H5 medium, and 100 μL was added per 10 mL of medium.

[0052] 1-11. Purification of VLPs The entire culture medium was centrifuged at 8,000 rpm for 10 min at 4°C to roughly remove the cells. This was followed by centrifugation at 11,000 rpm for 1 h at 4°C, and the supernatant was collected in a new centrifuge tube. This supernatant was transferred to a centrifuge tube (Beckman Coulter) and ultracentrifuged at 32,000 rpm for 2 h at 4°C using an Optima XPN-100 (Beckman Coulter) with an SW32-Ti rotor to precipitate proteins, including VLPs. The supernatant was discarded, and 1 mL of fresh H5 medium was added. The mixture was left to stand at 4°C for several hours to allow the pellet to swell. The swollen pellet was resuspended by pipetting until no precipitate remained. 1.8 g of CsCl (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed per tube, dissolved in 2 mL of H5 medium, and mixed with the pellet suspension. The mixture was placed in a centrifuge tube (Beckman Coulter) and ultracentrifuged at 40,000 rpm for 20 hours at 4°C using an SW55-Ti rotor. After centrifugation, white light was shone from above the tube, and only the VLP-derived band was collected using a pipette. The collected solution was transferred to a centrifuge tube for pelleting. The solution was made up to 1x PBS (Thermo Fisher Scientific) and ultracentrifuged at 32,000 rpm for 2 hours at 4°C to precipitate the VLPs. The supernatant was discarded, and fresh H5 medium was added. The mixture was left to stand overnight at 4°C to swell the pellet. The swollen pellet was suspended well by pipetting to obtain a purified VLP solution.

[0053] Quantification of VLPs The VLP concentration was quantified by the Bradford method. BSA diluted 2-fold from 0.5 mg / mL to 0.067 mg / mL was used as a standard. 200 μL of staining solution (Bio-Rad) was added to 800 μL of ion-exchanged water, and 20 μL of an appropriately diluted sample was added and mixed well. The mixture was left to stand at room temperature for 5 minutes, and the absorbance at 600 nm was measured using a spectrophotometer.

[0054] (Example 2) Selection of VHH by cDNA display method 2-1. Preparation of cDNA for VHH display Construction of a DNA library encoding the single variable domain of a heavy chain of a heavy-chain antibody (VHH) derived from a full-length alpaca antibody Using the S-hinge and L-hinge genes from two types of alpaca-derived naive VHH library provided by RePHAGEN as templates, the genes were amplified by PCR using an S-hinge VHH-specific primer pair (sequence numbers 114 and 115) and an L-hinge VHH-specific primer pair (sequence numbers 114 and 116). The resulting DNA fragments were then extended using overlapping PCR primers (sequence numbers 117 and 118) to prepare DNA fragments consisting of a T7 promoter, omega (ω) enhancer, Kozak consensus sequence, VHH gene, His tag, and linker hybridization region (Y tag), thereby creating a full-length VHH-encoding DNA library.

[0055] 2-2. Preparation of cDNA display Each cDNA display was prepared according to the following steps 2-3 to 2-7. The buffers used are listed in Table 9.

[0056] [Table 9]

[0057] 2-3. Transcription The full-length VHH-encoding DNA library prepared in 2-1 was transcribed using the T7 RiboMAX Express Large Scale RNA Production System (Promega) according to the attached manual. The amount of DNA used was 6.6 μg. The amount of DNA used for each subsequent selection was 0.1–1 μg from the second round onward. The resulting transcription product was purified using RNAClean XP (Beckman Coulter) according to the attached manual. The concentration of the purified product was quantified using a NanoPad DS-11FX (DeNovix).

[0058] 2-4. Ligation 20 pmol of purified mRNA and 20 pmol of the puromycin-linked cnvK riboG linker (Epsilon Molecular Engineering) were added to NaCl (final concentration 0.2 M) and Tris-HCl (pH 7.5, final concentration 0.05 M) and incubated at 90°C for 1 minute. The mixture was then cooled to 70°C at a rate of 0.1°C / sec and incubated at 70°C for 1 minute. The mixture was then cooled to 25°C at a rate of 0.1°C / sec and then to 10°C at a rate of 2°C / sec, allowing the cnvK linker to hybridize to the 3' end of the mRNA. Subsequently, a UVP CrossLinker (CL-3000), 365 nm, 100-115 V (Analytik Jena) was used to hybridize the cnvK linker to the 3' end of the mRNA. The resulting mixture was then irradiated with 4060 mJ / cm UV at 365 nm. 2 By irradiation, the cnvK linker and mRNA were photocrosslinked to obtain an mRNA-linker complex.

[0059] 2-5. Preparation of mRNA display Six pmol of the mRNA-linker complex was incubated at 30°C for 30 minutes in a 50-µL cell-free translation system (Rabbit reticulocyte lysate (nuclease-treated), Promega). MgCl2 and KCl were then added to final concentrations of 75 mM and 900 mM, respectively, and the mixture was incubated at 37°C for 1 hour to display the peptide corresponding to the mRNA on the puromycin residue on the mRNA-linker. EDTA (pH 8.0) was then added to a final concentration of 70 mM, and the mixture was incubated at 4°C for 5 minutes to prepare the mRNA display.

[0060] 2-6. Preparation of cDNA display 60 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific) was placed in a Protein Lobind tube and washed with 200 μL of binding buffer. The mRNA display prepared in methods 2-5 above was then added and stirred at 25°C for 30 minutes. After washing with 200 μL of binding buffer, the tube was incubated at 42°C for 30 minutes in a reaction solution with the composition shown in Table 10 for reverse transcription to prepare an mRNA / cDNA-VHH conjugate. After washing with 200 μL of binding buffer, 39 μL of His tag binding / washing buffer and 1 μL of 1,000 U / μL RNase T1 were added and stirred at 37°C for 15 minutes to elute the mRNA / cDNA-VHH conjugate (hereafter referred to as cDNA display) from Dynabeads Myone streptavidin C1 (Veritas).

[0061] [Table 10]

[0062] 2-7. Purification of cDNA display 30 μL of His Mag Sepharose Ni Beads (GE Health Care) were placed in an Eppendorf tube and washed with 200 μL of His tag binding / washing buffer. The cDNA display prepared in methods 2-1 to 2-6 was then added and stirred at 25°C for 30 minutes. After washing with 200 μL of His tag binding / washing buffer, 30 μL of His tag elution buffer was added and the cDNA display was eluted by stirring at 37°C for 15 minutes.

[0063] 2-8. In vitro selection experiments Selection was performed using cDNA display prepared from the library obtained in 2-7. The synthesis scale of cDNA display used in each selection round is shown in Table 11.

[0064] [Table 11]

[0065] 2-9. Screening procedure for selection cycle 1 (R1) 100 μL of 100 μg / mL GII.4 or GII.1 VLPs prepared with PBS was added to each well of an F96 Cert. Maxisorp Nunc-Immuno Plate (Thermo Fisher Scientific), sealed, and allowed to stand overnight at 4°C. After removing any unadsorbed norovirus VLP dispersion, 200 μL of PBST (PBS containing 0.05% (v / v) Tween 20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of 5% skim milk solution (PBST solvent) was added and incubated at room temperature for 1 hour. The 5% skim milk solution was then carefully removed using a pipette. 200 μL of HBST (20 mM HEPES, 500 mM NaCl, 0.05% Tween 20, pH 7.2) was added and immediately removed. This washing procedure was repeated three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs, and the mixture was incubated at room temperature for 1 hour. After this, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, allowed to stand at room temperature for 5 minutes, and then removed. This washing procedure was repeated six times. After washing, 100 μL of 100 mM Tris(hydroxymethyl)aminomethane (pH 11) prepared with nuclease-free water was added to each well. The mixture was carefully pipetted and allowed to stand at 37°C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLPs. The eluate was subjected to PCR using cnvK NewYtag for poly A (SEQ ID NO: 119) and T7 omeganew (SEQ ID NO: 120) as primers. The reaction mixture for each sample was prepared by mixing 25 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa Biosystems), 1.5 μL of 10 μM cnvK NewYtag for polyA primer, 1.5 μL of 10 μM T7 omeganew primer, 12.5 μL of VHH-presenting cDNA display, and 9.5 μL of nuclease-free water. PCR was performed at 95°C for 2 minutes, followed by 22 cycles of 98°C for 20 seconds, 68°C for 15 seconds, and 72°C for 20 seconds, followed by 72°C for 5 minutes. PCR products were purified using Agencourt AMPure XP (Beckman Coulter). The resulting purified DNA library (hereafter referred to as the R1 library) was subjected to R2 selection.

[0066] 2-10. Screening procedure for selection cycle 2 (R2) A cDNA display library was prepared from the R1 library using the same method as described above, at the synthesis scale shown in Table 3. 100 μL of 10 μg / mL GII.4 VLP or GII.1 VLP prepared with PBS was added to each well of an F96 Cert. Maxisorp Nunc-Immuno Plate (Thermo Fisher Scientific), sealed, and allowed to stand overnight at 4°C. After removing any norovirus VLP dispersion that had not adsorbed to the wells, 200 μL of PBST was added and immediately removed. This washing procedure was repeated three times. 200 μL of 5% skim milk solution (PBST solvent) was added and incubated at room temperature for 1 hour, after which the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST was added and immediately removed. This washing procedure was repeated three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs, and the mixture was incubated at room temperature for 1 hour. After this, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, allowed to stand at room temperature for 5 minutes, and then removed. This washing procedure was repeated six times. After washing, 100 μL of 100 mM Tris(hydroxymethyl)aminomethane (pH 11) prepared with nuclease-free water was added to each well. The mixture was carefully pipetted and then allowed to stand at 37°C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLPs. The eluate was subjected to PCR using cnvK NewYtag for poly A (SEQ ID NO: 119) and T7 omeganew (SEQ ID NO: 120) as primers. The reaction mixture for each sample was prepared by mixing 25 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa Biosystems), 1.5 μL of 10 μM cnvK NewYtag for polyA primer, 1.5 μL of 10 μM T7 omeganew primer, 12.5 μL of VHH-presenting cDNA display, and 9.5 μL of nuclease-free water. PCR was performed at 95°C for 2 minutes, followed by 24 cycles of 98°C for 20 seconds, 68°C for 15 seconds, and 72°C for 20 seconds, followed by 72°C for 5 minutes. PCR products were purified using Agencourt AMPure XP (Beckman Coulter). The resulting purified DNA library (hereafter referred to as the R2 library) was subjected to R3 selection.

[0067] 2-11. Screening procedure for selection cycles (R3 and R4) A cDNA display library was prepared from the R2 library using the same method as above, at the synthesis scale shown in Table 10. Screening was then carried out up to R4 according to the method in 2-10. The resulting purified DNA library (hereinafter referred to as the R4 library) was subjected to R5 selection.

[0068] 2-12. Screening procedure for selection cycle 5 (R5) A cDNA display library was prepared from the R4 library using the same method as described above, at the synthesis scale shown in Table 3. 100 μL of 1 μg / mL GII.4 VLP or GII.1 VLP prepared with PBS was added to each well of an F96 Cert. Maxisorp Nunc-Immuno™ Plate (Thermo Fisher Scientific), sealed, and allowed to stand overnight at 4°C. After removing the norovirus VLP dispersion that had not adsorbed to the wells, 200 μL of PBST was added and immediately removed. This washing procedure was repeated three times. 200 μL of 5% skim milk solution (PBST solvent) was added and incubated at room temperature for 1 hour, after which the 5% skim milk solution was carefully removed using a pipette. 200 μL of HBST was added and immediately removed. This washing procedure was repeated three times. 100 μL of the VHH-presenting cDNA display library prepared using HBST was added to each well containing immobilized VLPs, and the mixture was incubated at room temperature for 1 hour. After this, the unreacted VHH-presenting cDNA display library was carefully removed using a pipette. 200 μL of HBST was added, allowed to stand at room temperature for 5 minutes, and then removed. This washing procedure was repeated six times. After washing, 100 μL of 100 mM Tris(hydroxymethyl)aminomethane (pH 11) prepared with nuclease-free water was added to each well. The mixture was carefully pipetted and then allowed to stand at 37°C for 10 minutes to elute the VHH-presenting cDNA display bound to the immobilized VLPs. The eluate was subjected to PCR using cnvK NewYtag for poly A (SEQ ID NO: 119) and T7 omeganew (SEQ ID NO: 120) as primers. The reaction mixture for each sample was prepared by mixing 25 μL of KAPA HiFi HotStart Ready Mix (2X) (Kapa Biosystems), 1.5 μL of 10 μM cnvK NewYtag for poly A primer, 1.5 μL of 10 μM T7 omeganew primer, 12.5 μL of VHH-presenting cDNA display, and 9.5 μL of nuclease-free water. PCR was performed at 95°C for 2 minutes, followed by 24 cycles of 98°C for 20 seconds, 68°C for 15 seconds, and 72°C for 20 seconds, followed by 72°C for 5 minutes. PCR products were purified using Agencourt AMPure XP (Beckman Coulter). This resulted in the R5 library.

[0069] 2-13. Next-generation sequencer analysis To confirm the convergence of the R4 and R5 libraries in detail, we performed sequence analysis using a next-generation sequencer. Sequencing samples were prepared according to the 2-step PCR Amplicon Library Preparation method provided by Illumina. First, amplicon PCR was performed using the PCR products from each selection as templates and the NGS Fw 1st PCR primer (SEQ ID NO: 121) and NGS Rv 1st PCR primer (SEQ ID NO: 122). PCR conditions were 98°C for 1 minute, followed by 15 cycles of 98°C for 10 seconds, 62°C for 5 seconds, and 72°C for 35 seconds, followed by 72°C for 1 minute. The resulting PCR products were purified according to the Agencourt AMPure XP (Beckman Coulter) instructions, followed by index PCR according to the Illumina instructions. The resulting PCR product was purified according to the Agencourt AMPure XP instructions, and then its concentration was quantified using a NanoPad DS-11FX (DeNovix). Sequence analysis was then performed using MiSeq (Illumina) and MiSeq Reagent Nano kit v2 (500 cycles) according to the Illumina instructions.

[0070] 2-14.Sequence Data Analysis The demultiplexed sequence data was analyzed using a MiSeq Controller. The nucleotide sequence region encoding VHH was extracted from the sequence data and translated into amino acid sequences. The number of amino acid sequences that perfectly matched each amino acid sequence was then counted. As a result, NoVHH204 (SEQ ID NO: 20), NoVHH205 (SEQ ID NO: 21), and NoVHH26 (SEQ ID NO: 22) appeared frequently and were selected as antibodies with high affinity for the antigen.

[0071] In the amino acid sequence of NoVHH204 (sequence number 20), positions 1 to 26 are FR1 (sequence number 10), positions 27 to 36 are CDR1 (sequence number 1), positions 37 to 50 are FR2 (sequence number 13), positions 51 to 57 are CDR2 (sequence number 2), positions 58 to 96 are FR3 (sequence number 16), positions 97 to 100 are CDR3 (sequence number 3), and positions 101 to 111 are FR4 (sequence number 19).

[0072] In the amino acid sequence of NoVHH205 (sequence number 21), positions 1 to 26 are FR1 (sequence number 11), positions 27 to 36 are CDR1 (sequence number 4), positions 37 to 50 are FR2 (sequence number 14), positions 51 to 59 are CDR2 (sequence number 5), positions 60 to 98 are FR3 (sequence number 17), positions 99 to 112 are CDR3 (sequence number 6), and positions 113 to 123 are FR4 (sequence number 19).

[0073] In the amino acid sequence of NoVHH26 (sequence number 22), positions 1 to 26 are FR1 (sequence number 12), positions 27 to 36 are CDR1 (sequence number 7), positions 37 to 50 are FR2 (sequence number 15), positions 51 to 60 are CDR2 (sequence number 8), positions 61 to 99 are FR3 (sequence number 18), positions 100 to 106 are CDR3 (sequence number 9), and positions 107 to 117 are FR4 (sequence number 19).

[0074] (Example 3) Production of VHH using protease-deficient recombinant Bacillus subtilis 4-1. Artificial synthesis of genes The amino acid sequences of the synthesized VHHs were modified by adding a His tag to the C-terminus of the amino acid sequences of VHHs NoVHH204 (SEQ ID NO: 20), NoVHH205 (SEQ ID NO: 21), and NoVHH26 (SEQ ID NO: 22) via a linker sequence. The VHHs synthesized in this manner are referred to as His-tagged NoVHH204 (SEQ ID NO: 102), His-tagged NoVHH205 (SEQ ID NO: 103), and His-tagged NoVHH26 (SEQ ID NO: 104). These VHHs represented by SEQ ID NOs: 102 to 104 are collectively referred to as His-tagged VHHs. The artificial synthetic genes for synthesizing these His-tagged VHHs, the artificial synthetic gene for His-tagged NoVHH204 (sequence number 105), the artificial synthetic gene for His-tagged NoVHH205 (sequence number 106), and the artificial synthetic gene for His-tagged NoVHH26 (sequence number 107), were synthesized using Thermo Fisher Scientific's gene synthesis contract service and used in the experiments.

[0075] 4-2. Construction of a plasmid for expressing His-tagged VHH The plasmid sequence was amplified by PCR using the recombinant plasmid pHY-S237 (JP 2014-158430) constructed based on pHY300PLK as a template, a primer set of 5'-GATCCCCGGGAATTCCTGTTATAAAAAAAGG-3' (SEQ ID NO: 108) and 5'-ATGATGTTAAGAAAGAAAACAAAGCAG-3' (SEQ ID NO: 109), and PrimeSTAR Max DNA polymerase (TaKaRa). The promoter DNA derived from the spoVG gene was amplified by PCR using the genome of strain 168 as a template and a primer set of 5'-GAATTCCCGGGGATCTAAGAAAAGTGATTCTGGGAGAG-3' (SEQ ID NO: 110) and 5'-CTTTCTTAACATCATAGTAGTTCACCACCTTTTCCC-3' (SEQ ID NO: 111). The resulting promoter DNA was integrated into a plasmid sequence using the In-Fusion HD Cloning Kit (Takara) to construct a VHH expression plasmid linked to the spoVG promoter. The plasmid sequence was amplified by PCR using the primer set 5'-TGCTGCAAGAGCTGCCGGAAATAAA-3' (SEQ ID NO: 112) and 5'-TCTATTAAACTAGTTATAGGG-3' (SEQ ID NO: 113) and PrimeSTAR Max DNA polymerase (Takara). DNA containing the artificially synthesized genes was integrated into the resulting PCR fragment using the In-Fusion HD Cloning Kit (Takara), to construct a VHH expression plasmid containing each of the artificially synthesized VHH genes.

[0076] 4-3. Construction of recombinant Bacillus subtilis A strain lacking extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, and aprX) was created from Bacillus subtilis strain 168 (hereinafter referred to as strain 168) according to the method described in Japanese Patent No. 4,485,341. Furthermore, the sigF gene involved in sporulation was deleted from Bacillus subtilis strain Dpr9, which lacks all nine of the above extracellular protease genes, according to the method described in Japanese Patent No. 4,336,082. The resulting strain lacking multiple extracellular proteases is referred to as Dpr9ΔsigF. Plasmid introduction into the Dpr9ΔsigF strain was performed using the protoplast method described below. Glycerol-stocked B. subtilis was inoculated into 1 mL of LB liquid medium and cultured overnight at 30°C with shaking. The next day, 10 μL of this culture was inoculated into a fresh 1 mL of LB liquid medium and cultured at 37°C with shaking for approximately 2 hours. The culture was collected in a 1.5 mL tube and centrifuged at 12,000 rpm for 5 minutes. The supernatant was removed and the pellet was suspended in 500 μL of SMMP (0.5 M sucrose, 20 mM disodium maleate, 20 mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic Medium 3 (Difco)) containing 4 mg / mL Lysozyme (Sigma-Aldrich) and incubated at 37°C for 1 hour. The culture was then centrifuged at 3,500 rpm for 10 minutes. The supernatant was removed and the pellet was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with each plasmid, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to the mixture, mixed by inversion, and shaken at 30°C for 1 hour. The entire mixture was then spread onto a DM3 agar plate (1% CMC (Kanto Chemical), 0.5% Bacto™ Casamino Acids, 0.5% Bacto™ Yeast Extract, 8.1% disodium succinate 6H2O, 0.35% dipotassium hydrogen phosphate, 0.15% potassium dihydrogen phosphate, 0.5% glucose, 20 mM magnesium chloride, 0.01% BSA, 50 ppm tetracycline, 1% agar) and incubated at 30°C for 2–3 days.

[0077] 4-4.VHH production The recombinant B. subtilis prepared in 4-3 was inoculated into 1 mL of LB medium containing 15 ppm tetracycline and shaken overnight at 30°C to prepare a preculture. The preculture was then inoculated into 20 mL of 2xL-mal medium (2% Bacto™ TTryptone, 1% Bacto™ Yeast Extract, 1% sodium chloride, 7.5% maltose monohydrate, 7.5 ppm manganese sulfate, 15 ppm tetracycline) in a pleated Erlenmeyer flask at 1% concentration and cultured at 30°C for 72 hours with shaking. At the end of the culture, 1 mL of the culture was centrifuged in a microtube at 4°C, 15,000 rpm, and 5 minutes, and the supernatant was collected. His-tagged VHH was purified using Ni-NTA agarose beads (Fujifilm Wako Pure Chemical Industries, Ltd.) according to the kit's protocol. The purified protein was dissolved in PBS containing 30 mM imidazole.

[0078] Example 4: Evaluation of binding specificity by ELISA method 100 μL of 0 μg / mL or 10 μg / mL norovirus VLPs prepared with PBS were added to each well of an F96 Cert. Maxisorp Nunc-Immuno™ Plate (Thermo Fisher Scientific), sealed, and allowed to stand overnight at 4°C. After removing any norovirus VLP dispersion that had not adsorbed to the wells, 200 μL of PBST (PBS containing 0.05% (v / v) Tween 20) was added and immediately removed. This washing procedure was repeated three times. 200 μL of 5% skim milk solution (PBST solvent) was added, and the plate was incubated at room temperature for 1 hour. The 5% skim milk solution was then carefully removed using a pipette. 200 μL of PBST was added and immediately removed. This washing procedure was repeated three times. To each well containing immobilized VLPs, 100 μL of 2 μg / mL of each His-tagged VHH, prepared with PBST, was added and incubated at room temperature for 1 hour. Unreacted VHHs were then carefully removed using a pipette. 200 μL of PBST was added and immediately removed. This washing procedure was repeated three times. Anti-His-tag mAb-Biotin (Monoclonal, OGHis) (MEDICAL & BIOLOGICAL LABORATORIES) was used. The primary antibody was diluted 1 / 5,000 with PBST. 100 μL of primary antibody was added to each well and incubated at room temperature for 1 hour. After carefully removing the primary antibody using a pipette, 200 μL of PBST was added and then carefully removed using a pipette. This washing procedure was repeated three times. Streptavidin HRP Conjugate (Tokyo Chemical Industry Co., Ltd.) was used for primary antibody detection. Streptavidin-HRP conjugate was diluted 1 / 5,000 with PBST, and 100 μL of this was added to each well. The plate was then incubated at room temperature for 1 hour. After carefully removing the streptavidin-HRP conjugate with a pipette, 200 μL of PBST was added and carefully removed with a pipette. This washing procedure was repeated three times.The chromogenic substrate was prepared by dissolving OPD tablets (Thermo Fisher Scientific) in Stable Peroxide Substrate Buffer (Thermo Fisher Scientific). 100 μL of chromogenic substrate was added to each well, and after 30 minutes of incubation in the dark, absorbance was immediately measured at 450 nm using a Microplate Reader Infinite M1000 PRO (TECAN). The signal-to-noise (S / N) ratio was calculated by defining the value obtained from the wells with the antigen as the signal and the value obtained from the wells without the antigen as the noise.

[0079] The reactivity of each VHH was calculated by assigning an S / N ratio of less than 2 as "-," a ratio of 2 to less than 10 as "+," and a ratio of 10 or greater as "++." The binding activity of His-tagged VHHs to VLPs of each genotype is shown in Table 12. NoVHH204 and NoVHH26 were found to exhibit very broad reactivity with GI and GII human norovirus VLPs. In particular, NoVHH26 was found to strongly react with epidemic human norovirus strains GII.2, GII.4, and GII.17. NoVHH205 was also found to react with GII.3, GII.4, GII.10, and GII.22 human norovirus VLPs.

[0080] [Table 12]

Claims

1. An antibody that binds to GI and GII noroviruses, which has one or more structural domains containing CDRs shown in (a) or (b) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9).

2. The antibody of claim 1 , which binds to a norovirus structural protein.

3. The antibody according to claim 1 or 2, which is a single domain antibody or a multimer thereof.

4. The antibody of claim 3, wherein the single domain antibody is a VHH antibody.

5. The antibody according to claim 3, wherein the single domain antibody multimer is a multimer in which multiple structural domains are linked together.

6. The antibody according to claim 3, wherein the single domain antibody multimer is a multimer in which one or more of the structural domains are linked to one or more structural domains having different antigen specificity from the structural domains.

7. A nucleic acid encoding the antibody according to any one of claims 1 to 6.

8. An antibody mixture that binds to GI and GII type noroviruses, comprising one or more antibodies selected from those that bind to noroviruses and have one or more structural domains containing CDRs shown in (a) and (b) below, and an antibody that binds to noroviruses and has one or more structural domains containing CDRs shown in (c) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by GFSVNSLTMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GMNKDGSTS (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by QGPLFRPDLPLYDS (SEQ ID NO: 6).

9. A method for detecting human norovirus in a sample, comprising the step of contacting a test sample with an antibody that binds to GI and GII noroviruses and has one or more structural domains containing CDRs shown in (a) or (b) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9).

10. A method for detecting human norovirus in a sample, comprising the step of contacting a test sample with a mixture of antibodies that bind to GI and GII noroviruses, the mixture including one or more antibodies selected from those that bind to noroviruses having one or more structural domains containing CDRs shown in (a) and (b) below, and antibodies that bind to noroviruses having one or more structural domains containing CDRs shown in (c) below. (a) CDR1 consisting of the amino acid sequence represented by GRTFSMYTMG (SEQ ID NO: 1), CDR2 consisting of the amino acid sequence represented by AISWRTY (SEQ ID NO: 2), and CDR3 consisting of the amino acid sequence represented by DGGY (SEQ ID NO: 3). (b) CDR1 consisting of the amino acid sequence represented by GFTFSHAYMS (SEQ ID NO: 7), CDR2 consisting of the amino acid sequence represented by AINYSGDSTF (SEQ ID NO: 8), and CDR3 consisting of the amino acid sequence represented by FWWTYDY (SEQ ID NO: 9). (c) CDR1 consisting of the amino acid sequence represented by GFSVNSLTMS (SEQ ID NO: 4), CDR2 consisting of the amino acid sequence represented by GMNKDGSTS (SEQ ID NO: 5), and CDR3 consisting of the amino acid sequence represented by QGPLFRPDLPLYDS (SEQ ID NO: 6).

11. A human norovirus detection kit comprising the antibody according to any one of claims 1 to 6 or the antibody mixture according to claim 8.

12. A preventive or therapeutic agent for human norovirus infection, comprising the antibody according to any one of claims 1 to 6 or the antibody mixture according to claim 8.

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