WO2020036135A1 - Human norovirus-like particle and use thereof - Google Patents

Abstract

The present invention addresses the problem of providing an antigen for generating an antibody for inhibiting infection of multiple types of HuNov, a use thereof, and an antibody for inhibiting infection of multiple types of HuNov. Specifically, the present invention provides: a method for producing an antibody for inhibiting infection of different genotypes of human norovirus (HuNoV) to intestinal epithelial cells, comprising immunization using the full length or a part of VP1 protein of GII.17 HuNoV as an antigen; and an antibody for inhibiting infection of a plurality of different genotypes of HuNoV to intestinal epithelial cells.

Description

Human norovirus-like particles and uses thereof

The present invention relates to human norovirus-like particles and uses thereof.

Human norovirus (HuNoV) is a pathogenic virus that invades cells of the upper small intestine from the duodenum and causes nonbacterial acute gastroenteritis, and belongs to the non-enveloped Calicivirus family.
The HuNoV genome is a single-stranded plus-sense RNA of about 7.7 kb. A viral protein called VPg is covalently linked to the 5 'end of the genome and the 3' end is polyadenylated. In the genome, there are three protein coding regions, ORF1, VP1 and VP2, which code for non-structural protein, structural protein 1 and structural protein 2, respectively. Structural analysis reveals that HuNoV constructs a truncated icosahedral structure with 90 (by 180 molecules) VP1 dimers. Each VP1 monomer is divided into two domains, a shell domain (S domain) and a protruding domain (P domain), of which the P domain is further divided into P1 and P2 subdomains. P2 recognizes blood group antigens that function as receptors and host infectious agents during infection. Mutations in the P2 subdomain cause changes in binding to blood group antigens (Non-Patent Document 1).

HuNoV is classified into three gene groups (GI, GII and GIV) based on the genome sequence, and it is said that there are at least 25 genotypes. Although it is a norovirus that shows high diversity, in recent years, only a small number of strains such as GII, particularly genotype 4 (GII.4) have been responsible for the spread of infection (Non-Patent Document 1).
About 20 million HuNoV infections are transmitted annually in the U.S., and about 70,000 are hospitalized. In Japan, there is no clear data, but about 1 to 20,000 inpatients and more than 7 million infections Are estimated to occur every year. Most of HuNoV infections have been relieved, but some children and the elderly have died.Severe cases have occurred, and outbreaks have often occurred in facilities such as kindergartens and nursing homes, and the number of outpatients and inpatients is considerable. And the burden on the social insurance system is large.

To date, several HuNoV vaccine candidates, including the GII.4 virus like particle (VLP), have been developed and are undergoing preclinical and clinical trials. VLPs are considered to be good vaccine antigen candidates because they have almost the same outer shell structure and antigenicity as live infectious viruses (Non-Patent Document 2). GII.4 is the main genotype in the HuNoV outbreak, but recently other genotypes, especially GII.2, GII.3, GII.6 and GII.17, have become more influential (non-patented). References 3-6). Therefore, there is a need for the development of viral therapeutics such as cross-reactive antibodies that inhibit viral infection of several genotypes.
However, at present, there is no antibody that inhibits viral infection of multiple genotypes or a single vaccine antigen that can induce it.

In view of the above circumstances, an object of the present invention is to provide an antigen for producing an antibody that suppresses multiple types of HuNoV infection, use thereof, and an antibody that suppresses multiple types of HuNoV infection.

The present inventors have generated antibodies against GII.4 VLP, GII.3 VLP, and GII.17 VLP, and used a line of intestinal epithelial cells derived from iPS cells (iPSCs) to generate several genes for HuNoV. The type was tested to determine whether it inhibited its infection (HuNoV invasion into and / or replication within intestinal epithelial cells).
As a result, an antibody prepared using VLPs formed by self-aggregation of the VP1 protein of GII.4 as an antigen was able to suppress the replication of GII.4, but suppressed the replication of HuNoV of other genotypes. Did not. Antibodies prepared using VLPs formed by self-aggregation of the VP1 protein of GII.3 as antigens were able to suppress the replication of GII.3, but inhibited the replication of HuNoV of other genotypes. Did not.
In contrast, an antibody prepared using VLPs formed by self-aggregation of the VP1 protein of GII.17 as antigens not only suppresses GII.17 replication but also effectively suppresses GII.4 replication. I understood.
Based on the above findings, the present invention provides an antigen suitable for the purpose of producing an antibody that inhibits HuNoV infection of a plurality of different genotypes (particularly, GII.4 HuNoV and GII.17 HuNoV), and an antigen produced using the antibody. And the like.

More specifically, the present invention includes the following (1) to (10).
(1) A method for producing an antibody that inhibits infection of human norovirus (HuNoV) of a plurality of different genotypes into intestinal epithelial cells, comprising immunizing with the full length or a part of the VP1 protein of GII.17 HuNoV as an antigen. A method for producing an antibody, comprising:
(2) The method for producing an antibody according to (1) above, wherein the full length of the VP1 protein of GII.17 HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
(3) The method for producing an antibody according to the above (1) or (2), wherein the plurality of different genotypes are GII.4 and GII.17.
(4) An antibody that binds to the VP1 protein of GII.17 HuNoV, and inhibits infection of multiple different genotypes of HuNoV into intestinal epithelial cells or a functional fragment thereof.
(5) The antibody or the functional fragment thereof according to (4), wherein the full length of the VP1 protein of GII.17 HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
(6) The antibody or the functional fragment thereof according to the above (4) or (5), wherein the plurality of different genotypes are GII.4 and GII.17.
(7) A pharmaceutical composition comprising the antibody or the functional fragment thereof according to any of (4) to (6).
(8) A vaccine for suppressing infection of HuNoV of a plurality of different genotypes, which contains the full length or part of GII.17 HuNoV VP1 protein as an antigen.
(9) The vaccine according to the above (8), wherein the full length of the VP1 protein of GII.17 HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
(10) The vaccine according to (8) or (9), wherein the plurality of different genotypes are GII.4 and GII.17.

The present invention provides an antigen for producing an antibody that suppresses the replication of HuNoV of a plurality of different genotypes (for example, GII.4 and GII.17).

The present invention also provides an antibody against the above antigen. The antibody can suppress HuNoV infection of a plurality of different genotypes (eg, GII.4 and GII.17), and can provide an effective treatment for HuNoV infection.

Effect of anti-GII.4 VLP polyclonal antibody on HuNoV replication. A is the genomic copy number of GII.4 HuNoV (strain 17-231) cultured after pretreatment with anti-GII.4 VLP polyclonal antibody, B is GII. 3 shows the results of measuring the genome copy number of GII.17 and GII.6 HuNoV. Measurements were taken at 3 hours (open) and 72 hours (black) after infection. As a control IgG, an unimmunized rabbit-derived IgG was used. Each value is a representative value of three independent experiments and is shown as a mean value ± standard deviation (SD). Effect of anti-GII.3 VLP polyclonal antibody on HuNoV replication. A is the genomic copy number of GII.3 HuNoV cultured after pretreatment with an anti-GII.3 VLP polyclonal antibody, B is GII.4 (17B93 strain), which was pretreated and cultured with an anti-GII.3 VLP polyclonal antibody, It is the result of measuring the genome copy number of GII.17 and GII.6 HuNoV. Measurements were taken at 3 hours (open) and 72 hours (black) after infection. As a control IgG, an unimmunized rabbit-derived IgG was used. Each value is a representative value of three independent experiments and is shown as a mean value ± standard deviation (SD). Effect of anti-GII.17 VLP polyclonal antibody on HuNoV replication. A shows the genomic copy number of GII.17 HuNoV cultured after pretreatment with anti-GII.17 VLP polyclonal antibody, and B shows GII.4 (strain 17-231) cultured after pretreatment with anti-GII.17 VLP polyclonal antibody. And 17B93 strain), and the results of measuring the genome copy number of GII.3 and GII.6 HuNoV. Measurements were taken at 3 hours (open) and 72 hours (black) after infection. As a control IgG, an unimmunized rabbit-derived IgG was used. Each value is representative of three independent experiments and is shown as the mean ± SD.

A first embodiment of the present invention relates to a method for producing an antibody that inhibits infection of a plurality of different genotypes of HuNoV into intestinal epithelial cells, the method comprising: This is a method for producing an antibody, including immunization as an antigen.
The antibody production method according to the first embodiment (hereinafter also referred to as “the antibody production method of the present invention”) shows cross-reactivity with HuNoV of different genotypes and inhibits their infection (or A method for producing an antibody that suppresses the VP1 protein, wherein the full length or a part of the VP1 protein of GII.17 HuNoV is used as an antigen. The genotype of HuNoV that the antibody produced by the antibody production method of the present invention inhibits infection is preferably GII.4 or GII.17 belonging to the GII genogroup.
In all embodiments of the present invention, the VP1 protein of the antigen GII.17 HuNoV may be its full length, or may be a partial region having antigenicity. It is a protein represented by SEQ ID NO: 3. The antigenic region can be easily selected by preparing a part of the VP1 protein of GII.17 HuNoV and using it as an immunogen to confirm the neutralizing ability of the antibody (the ability to suppress the replication of HuNoV) can do.

In all the embodiments of the present invention, “GII.17 HuNoV VP1 protein” refers to a protein including the amino acid sequence represented by SEQ ID NO: 3 as well as the amino acid sequence represented by SEQ ID NO: 3 And a protein comprising an amino acid sequence substantially identical to
Here, the term "protein containing substantially the same amino acid sequence" refers to the amino acid sequence represented by SEQ ID NO: 3 by about 60% or more, preferably about 70% or more, more preferably about 80%, 81%, 82% or more. %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Most preferably, the antibody against the protein comprises an amino acid sequence having about 99% amino acid identity and is capable of infecting intestinal epithelial cells with HuNoVs (eg, GII.4 and GII.17) of multiple different genotypes. Is a protein that has the ability to inhibit

Alternatively, a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3 refers to one or several (preferably about 1 to 30, more preferably Is composed of an amino acid sequence in which about 1 to 10, more preferably 1 to 5) amino acids have been deleted, substituted, inserted or added, and an antibody against the protein is intestinal tract of HuNoV of a plurality of different genotypes. A protein that has the ability to inhibit infection of epithelial cells.

The VP1 protein of GII.17 HuNoV is obtained by obtaining a nucleic acid (for example, SEQ ID NO: 4 or the like) encoding this protein from a cDNA library or the like, incorporating the nucleic acid into an appropriate expression vector, and transforming an appropriate host cell with the expression vector. It can be prepared by transforming or transfecting, culturing this in an appropriate medium, expressing and purifying the VP1 protein.

Examples of host cells for expressing the VP1 protein include bacterial cells (eg, Escherichia coli B strain , E. coli Kl2 strain, Corynebacterium ammoniagenes , C. glutamicum , Serratia liquefaciens , Streptomyces lividans , Pseudomonas putida, etc.); molds (eg, Penicillium ) camembertii , Acremonium chrysogenum, etc.), animal cells, plant cells, baculovirus / insect cells or yeast cells (eg, Saccharomyces cerevisiae and Pichia pastoris ) can be used and expressed in these cells.

As an expression vector for expressing the VP1 protein, a vector suitable for various host cells can be used. Examples of expression vectors include, for example, pBR322, pBR325, pUC118, pET etc. (Escherichia coli host), pEGF-C, pEGF-N etc. (animal cell host), pVL1392, pVL1393 etc. (insect cell host, baculovirus vector), pG -1, Yep13 or pPICZ (yeast cell host) can be used. These expression vectors have a replication origin, a selection marker, and a promoter suitable for each vector, and may include an enhancer, a transcription assembly sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like, if necessary. You may have. Furthermore, in order to facilitate purification of the expressed polypeptide, a base sequence for expressing by fusion of a FLAG tag, a His tag, an HA tag, a GST tag and the like may be inserted into the expression vector.
Preparation of the expression vector can be carried out by a method known to those skilled in the art, and can also be carried out using a commercially available kit or the like as appropriate.

When extracting the expressed VP1 protein from cultured cells or cultured cells, after culturing, the cells or cultured cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme and / or lysozyme. Alternatively, after the cells or cells are destroyed by freeze-thawing or the like, a soluble extract is obtained by centrifugation or filtration. In particular, when using cultured cells as a host, it is desirable to obtain the VP1 protein expressed in the culture supernatant by collecting the supernatant. From the obtained extract or culture supernatant, a desired protein can be obtained by appropriately combining known separation and purification methods. Known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, methods using mainly differences in molecular weight such as SDS-PAGE, and ionization. A method using a charge difference such as exchange chromatography, a method using a specific affinity such as affinity chromatography (for example, when a polypeptide is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used. When expressing the polypeptide with the His tag, Ni-NTA resin or Co-based resin, when expressing the polypeptide with the HA tag, expressing the anti-HA antibody resin, expressing the polypeptide with the FLAG tag. In this case, a method using an anti-FLAG antibody binding resin, etc.), a method utilizing the difference in hydrophobicity such as reversed-phase high performance liquid chromatography, or isoelectric focusing method A method utilizing the difference between the electric points is used.

The antibody produced in the first embodiment is not particularly limited, and may be, for example, a monoclonal antibody, a polyclonal antibody, a nanoantibody, or the like.
When the antibody produced in the first embodiment is a polyclonal antibody, for example, an antigen can be used for an immunized animal (for example, but not limited to, rabbit, goat, sheep, chicken, guinea pig, mouse, rat or pig). And an adjuvant mixture can be prepared. Usually, the antigen and / or adjuvant is injected multiple times into the immunized animal subcutaneously or intraperitoneally. Adjuvants include, but are not limited to, for example, complete Freund and monophosphoryl lipid A synthesis-trehalose dicorynomycolate (MPL-TMD). After immunization with the antigen, serum containing an antibody against VP1 is prepared from the immunized animal, and the desired antibody can be purified by a conventional method (for example, a method using rProtein A-Sepharose).

When the antibody produced in the first embodiment is a monoclonal antibody, it can be produced as follows.
As used herein, the term “monoclonal” refers to a property of an antibody obtained from a substantially homogeneous population of antibodies, and the antibody is produced by a specific method (eg, a hybridoma method). It does not mean to be done.
Examples of a method for producing a monoclonal antibody include a hybridoma method (Kohler and Milstein, Nature 256 495 1975) or a recombinant method (US Pat. No. 4,816,567). Moreover, the monoclonal antibody according to the present invention may be isolated from a phage antibody library (Clackson et al., Nature 352 624-628 1991; Marks et al., J. Mol. Biol. 222 581-597 1991). . More specifically, when prepared using the hybridoma method, the preparation method includes, for example, the following four steps: (i) immunizing the VP1 protein of GII.17 HuNoV or a part thereof; Immunize animals, (ii) collect lymphocytes secreting (or potentially secreting) monoclonal antibodies, (iii) fuse lymphocytes with immortalized cells, (iv) secrete desired monoclonal antibodies Select cells. As the immunized animal, for example, mouse, rat, guinea pig, hamster and the like can be selected. After immunization, lymphocytes obtained from the host animal are fused with an immortalized cell line using a fusion agent such as polyethylene glycol to establish hybridoma cells. As the fused cells, for example, rat or mouse myeloma cell lines are used. After performing the cell fusion, the cells are grown in a suitable medium containing unfused lymphocytes and one or more substrates that inhibit the growth or survival of the immortalized cell line. The usual technique uses parental cells that lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT). In this case, hypoxanthine, aminopterin and thymidine inhibit the growth of HGPRT-deficient cells and are added to a medium that allows hybridoma growth (HAT medium). From the hybridomas thus obtained, a hybridoma producing a desired antibody is selected, and the desired monoclonal antibody can be obtained from a medium in which the selected hybridoma grows according to a conventional method.
The hybridoma thus prepared can be cultured in vitro or in vivo in ascites of mice, rats, guinea pigs, hamsters, etc., and the target antibody can be prepared from the culture supernatant or ascites.

A nanoantibody is a polypeptide comprising a variable domain of the heavy chain of an antibody (VHH). Usually, antibodies such as humans are composed of heavy chains and light chains. Camelid animals such as llamas, alpacas and camels produce single-chain antibodies consisting only of heavy chains (heavy chain antibodies). A heavy chain antibody can recognize a target antigen and bind to the antigen, similarly to a normal heavy and light chain antibody. The variable region of a heavy chain antibody is the smallest unit that has binding affinity for an antigen, and this variable region fragment is called a "nanoantibody." Nanoantibodies have high heat resistance, digestion resistance, and room temperature stability, and can be easily prepared in large quantities by genetic engineering techniques.
Nanoantibodies can be prepared, for example, as follows. A camelid animal is immunized with an antigen, the presence or absence of the target antibody is detected from the collected serum, and cDNA is prepared from RNA derived from peripheral blood lymphocytes of the immunized animal in which the desired antibody titer has been detected. A VHH-encoding DNA fragment is amplified from the obtained cDNA and inserted into a phagemid to prepare a VHH phagemid library. A desired nanoantibody can be prepared from the prepared VHH phagemid library through several screenings.

A second embodiment of the present invention relates to an antibody that binds to the VP1 protein of GII.17 HuNoV, wherein the antibody inhibits infection of multiple different genotypes of HuNoV into intestinal epithelial cells (hereinafter referred to as “the antibody of the present invention Or a functional fragment thereof.
As described above, the antibody of the present invention is not particularly limited, and may be, for example, a monoclonal antibody, a polyclonal antibody, or a nanoantibody. Further, the antibody of the present invention may be a recombinant antibody. The recombinant antibody is not particularly limited, and includes, for example, chimeric antibodies (for example, humanized antibodies and human antibodies). Here, a chimeric antibody is an antibody in which a variable region and a constant region derived from different animal species are linked, for example, an antibody in which the variable region of a mouse-derived antibody is linked to a human-derived constant region. Such linked antibodies can easily be constructed by genetic recombination techniques well known to those skilled in the art.
In addition, the genotype of HuNoV that the antibody of the present invention inhibits infection is preferably GII.4 and GII.17.

A functional fragment of the antibody of the present invention is a partial region of the antibody of the present invention, which means an antibody fragment that binds to HuNoV VPL and inhibits its infection or proliferation in intestinal epithelial cells, for example, Fab, Fab ', F (ab') 2, Fv (variable fragment of antibody), single chain antibody (heavy chain, light chain, heavy chain variable region, light chain variable region, nano antibody, etc.), scFv (single chain) Fv), diabody (scFv dimer), dsFv (disulfide-stabilized ΔFv), and peptides containing at least a part of the CDR of the antibody of the present invention.

Fab is an antibody fragment having antigen-binding activity in which about half of the N-terminal side of the heavy chain and the entire light chain are bound by disulfide bonds, among fragments obtained by treating an antibody molecule with the protease, papain. Fab is prepared by treating the antibody molecule with papain to obtain a fragment, for example, constructing an appropriate expression vector into which DNA encoding Fab is inserted, and inserting it into an appropriate host cell (for example, CHO cell or the like). After introduction into mammalian cells, yeast cells, insect cells, and the like), and then expressing Fab in the cells.

F (ab ') 2 is an antibody fragment having an antigen-binding activity, which is a little larger than a fragment obtained by treating an antibody molecule with the protease pepsin, wherein the Fab is bound via a disulfide bond in the hinge region. It is. F (ab ') 2 can be prepared by treating the antibody molecule with pepsin to obtain a fragment, or by linking the Fab to a thioether bond or a disulfide bond. can do.

'Fab' is an antibody fragment having antigen-binding activity, which is obtained by cleaving a disulfide bond in the hinge region of F (ab ') 2. Fab 'can also be produced by a genetic engineering technique in the same manner as Fab and the like.

scFv is a VH-linker-VL or VL-linker-VH polypeptide in which one heavy chain variable region (VH) and one light chain variable region (VL) are linked using an appropriate peptide linker. And an antibody fragment having an antigen-binding activity. The scFv can be prepared by obtaining cDNAs encoding the heavy chain variable region and the light chain variable region of the antibody, and using a genetic engineering technique.

Diabody is an antibody fragment obtained by dimerizing scFv and has a bivalent antigen-binding activity. The divalent antigen-binding activities may be the same antigen-binding activity or one may be a different antigen-binding activity. The diabody obtains cDNAs encoding the heavy chain variable region and light chain variable region of the antibody, constructs a cDNA expressing the scFv in which the heavy chain variable region and the light chain variable region are linked by a peptide linker, and performs genetic engineering. It can be produced by a technique.

DsFv refers to a polypeptide in which one amino acid residue in each of the heavy chain variable region and the light chain variable region has been substituted with a cysteine residue, which is linked via a disulfide bond between the cysteine residues. The amino acid residue to be substituted for the cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody. dsFv can be prepared by obtaining cDNAs encoding the heavy chain variable region and the light chain variable region of an antibody, constructing a DNA encoding the dsFv, and performing genetic engineering techniques.

The peptide containing the CDR is configured to include at least one region or more of the heavy chain or light chain CDRs (CDR1 to 3). Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker. For the peptide containing the CDR, a DNA encoding the heavy chain or light chain CDR of the antibody is constructed and inserted into an expression vector. The type of the vector is not particularly limited, and may be appropriately selected depending on the type of the host cell to be subsequently introduced. These can be produced by introducing them into an appropriate host cell (eg, a mammalian cell such as a CHO cell, a yeast cell, an insect cell, etc.) in order to express them as an antibody. Further, the peptide containing CDR can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method).

第 The third embodiment of the present invention is a medicine containing the antibody of the present invention or a functional fragment thereof (hereinafter, also referred to as “the medicine of the present invention”). The medicament of the present invention has a pharmacological effect as a prophylactic and / or therapeutic agent for infections caused by HuNoV. The medicament of the present invention may be in a form in which the antibody of the present invention as an active ingredient or the functional fragment thereof itself is administered, but generally, in addition to the antibody of the present invention as an active ingredient or the functional fragment thereof, Alternatively, it is desirable to administer in the form of a pharmaceutical composition containing two or more pharmaceutical additives (hereinafter also referred to as “the pharmaceutical composition of the present invention”). Further, the pharmaceutical composition according to the embodiment of the present invention may further contain other known drugs.

The medicament or the pharmaceutical composition according to the embodiment of the present invention is not particularly limited, and may be in the form of a tablet, capsule, granule, powder, syrup, suspension, suppository, ointment, cream, gel. Preparations, patches, inhalants or injections. These preparations are prepared according to a conventional method. In the case of liquid preparations, they may be dissolved or suspended in water or another suitable solvent at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the antibody of the present invention or a functional fragment thereof is prepared by dissolving the same in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, An agent may be added.

製 剤 Formulations for oral or parenteral administration are provided in any formulation form. Formulation forms include, for example, oral administration agents in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or solutions, intravenous administration, intramuscular It can be prepared in the form of injections, drops, transdermal absorbents, transmucosal absorbents, nasal drops, inhalants or suppositories for administration or subcutaneous administration. Injections, drops, and the like can be prepared as a powdery dosage form such as a lyophilized form, and dissolved in an appropriate aqueous medium such as physiological saline before use.

The type of the pharmaceutical additive used in the production of the medicament or the pharmaceutical composition according to the embodiment of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method of producing the medicament or the pharmaceutical composition depends on the form. Thus, a person skilled in the art can select as appropriate. Inorganic or organic substances, or solid or liquid substances can be used as pharmaceutical additives. Generally, for example, 0.1% to 99.9% by weight, 1% to 1% by weight based on the weight of the active ingredient. 95.0% by weight, or between 1% and 90.0% by weight. Specifically, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium as examples of pharmaceutical additives , Ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysodium Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol or water and the like.

To produce a solid preparation for oral administration, the active ingredient and excipient components, for example, lactose, starch, microcrystalline cellulose, calcium lactate or silicic acid anhydride and the like, or a powder, or A binder such as sucrose, hydroxypropylcellulose or polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose or calcium carboxymethylcellulose are added, and the mixture is granulated by wet or dry granulation to give granules. In order to produce tablets, these powders and granules may be compressed as they are or by adding a lubricant such as magnesium stearate or talc. These granules or tablets are coated with an enteric base such as hydroxypropylmethylcellulose phthalate or methacrylic acid-methyl methacrylate polymer to form an enteric coated preparation, or coated with ethyl cellulose, carnauba wax or hardened oil to obtain a sustained release preparation. You can also. To produce capsules, powders or granules are filled in hard capsules, or the active ingredient is used as it is, or is dissolved in glycerin, polyethylene glycol, sesame oil or olive oil, and then coated with gelatin to form soft capsules. be able to.

In order to manufacture an injection, the active ingredient may be used as necessary, such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, a pH adjuster such as sodium monohydrogen phosphate or sodium dihydrogen phosphate, sodium chloride or glucose, etc. Dissolve in distilled water for injection together with an isotonic agent, and aseptically filter and fill into ampoules, or add mannitol, dextrin, cyclodextrin or gelatin, and freeze-dry in vacuo to obtain a working-soluble injection. Is also good. In addition, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil or the like may be added to the active ingredient and emulsified in water to prepare an injection emulsion.

In order to manufacture a rectal preparation, the active ingredient is dissolved by humidification together with a suppository base such as cocoa butter, fatty acid tri-, di- and monoglycerides or polyethylene glycol, and then poured into a mold and cooled, or the active ingredient is cooled. After dissolving in polyethylene glycol or soybean oil or the like, it may be coated with a gelatin film.

The dose and the number of times of administration of the medicament or the pharmaceutical composition according to the embodiment of the present invention are not particularly limited, and the purpose of preventing and / or treating the deterioration and progression of the target disease, the type of the disease, the weight and age of the patient, and the like Can be appropriately selected by the judgment of the doctor or pharmacist according to the conditions of
In general, the daily dose for oral administration in adults is about 0.01 to 1,000 mg (weight of active ingredient), and it can be administered once or several times a day or every few days . When used as an injection, it is desirable to continuously or intermittently administer a daily dose of 0.001 to 100 mg (weight of the active ingredient) to an adult.

The medicament or the pharmaceutical composition according to the embodiment of the present invention is prepared as a sustained-release preparation such as an implant and a delivery system encapsulated in microcapsules using a carrier capable of preventing immediate removal from the body. can do. As such carriers, biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acids can be used. Such materials can be easily prepared by those skilled in the art. Also, a suspension of liposomes can be used as a pharmaceutically acceptable carrier. Liposomes are prepared through a filter of appropriate pore size to a size suitable for use as a lipid composition comprising, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE). Can be purified by

The medicament or the pharmaceutical composition according to the embodiment of the present invention may be provided in the form of a kit together with instructions such as an administration method. The medicament or the pharmaceutical composition contained in the kit is manufactured from a material that maintains the activity of the active ingredient effectively for a long period of time, does not cause the agent or the like to adsorb to the inside of the container, and does not alter the components. Supplied by container. For example, a sealed glass ampoule may include a buffer or the like encapsulated in the presence of a neutral, non-reactive gas such as nitrogen gas.
The kit may be accompanied by instructions for use. Instructions for use of the kit may be printed on paper or the like, or may be stored and supplied on an electromagnetically readable medium such as a CD-ROM or DVD-ROM.

A fourth embodiment of the present invention provides a vaccine for suppressing infection of a plurality of different genotypes of HuNoV, which contains the full length or a part of the VP1 protein of GII.17 HuNoV as an antigen (hereinafter referred to as “the vaccine of the present invention”). ").
The vaccine of the present invention may comprise one or more adjuvants, such as complete Freund's or incomplete Freund's adjuvant, cholera toxin, heat-labile Escherichia coli toxin, aluminum hydroxide, potassium alum, saponin or a derivative thereof, muramyl dipeptide, mineral oil or Vegetable oils, novasomes or non-ionic block copolymers, DEAE dextran and the like can be included. It may also contain a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier must be a compound that does not adversely affect the health of the animal being vaccinated. A pharmaceutically acceptable carrier is, for example, sterile water or a buffer.
The vaccine of the present invention can be administered by a conventional active immunization method, and may be administered by injection, orally, or by a transmucosal method such as nasal. In addition, the vaccine preparation of the present invention can be administered in a single dose by a method suitable for the dosage form in an amount effective for preventing or treating HuNoV infection (an amount sufficient to induce immunity in animals against HuNoV challenge). Or it can be administered multiple times. The vaccine can be administered intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally, or mucosally, such as intranasally or sublingually. In addition, the vaccine preparation of the present invention can be used by mixing with other antigen components.
The dose and frequency of administration of the vaccine preparation may vary depending on the administration subject, but protective immunity can be induced by administering the vaccine containing several tens of μg of the antigen once every several weeks, once every several weeks.

A fifth embodiment of the present invention comprises administering to a patient a medicament or pharmaceutical composition of the present invention or a vaccine of the present invention, wherein a plurality of different genotypes of HuNoV (eg, GII.4 and GII.17). For preventing and / or treating infectious diseases.
Here, `` treatment '' refers to preventing or alleviating the progress and worsening of the disease state in a patient already infected with HuNoV, and thereby preventing or alleviating the progress and worsening of HuNoV infection. Is the action to be taken.
In addition, `` prevention '' means, for a patient who may be infected with HuNoV, preventing the infection in advance, and thereby a treatment aimed at preventing the onset of HuNoV infection in advance. .

The disclosures of all documents cited herein are hereby incorporated by reference in their entirety. Also, when the specification is translated into English and the singular forms “a”, “an”, and “the” are included, the context does not clearly indicate otherwise. As long as it is not limited to one, it includes a plurality.
Hereinafter, the present invention will be further described with reference to examples. However, the examples are merely examples of the embodiments of the present invention, and do not limit the scope of the present invention.

Materials and methods Preparation of VLP and polyclonal antibody against VLP The virus was roughly purified from stool containing HuNoV donated by Osaka National Institute of Health and Safety, and a virus genome was prepared therefrom. Primers were set outside the VP1 ORFs of GII.4, GII.3 and GII.17 on the prepared genome, each ORF region was amplified by PCR, and the nucleotide sequence of the amplified product was determined. The ORF of each VP1 was cloned into pFastBac Dual Expression Vector (Invitrogen). The amino acid sequence of GII.4 VP1 and the nucleic acid sequence encoding the same are shown in SEQ ID NOS: 1 and 2, respectively, and the amino acid sequence of GII.17 VP1 and the nucleic acid sequence encoding the same are shown in SEQ ID NOs: 3 and 4, respectively. The amino acid sequence of GII.3 VP1 and the nucleic acid sequence encoding it are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
After confirming that the sequence was correct, each construct was used for producing a recombinant baculovirus of the Bac-to-Bac expression system (Invitrogen). High Five cells (Invitrogen) were infected with each recombinant baculovirus at a MOI (multiplicity of infection) of 7 pfu (plaque-forming units) / cell. Six days after infection, the culture supernatant was collected and centrifuged at 20,000 g for 1 hour. The obtained supernatant was ultracentrifuged at 100,000 g for 2 hours, and the precipitated VLP was suspended in PBS. The concentrated VLPs were layered on a 10% -60% sucrose density gradient and purified by ultracentrifugation at 100,000 g for 1 hour. VLPs passed through the sucrose density gradient were dialyzed three times against 2 L of PBS to remove sucrose in the sample. VLP was concentrated with AmiconUltra 30-kDa centrifugal filter (Millipore).
To prepare anti-HuNoV polyclonal antibodies, two rabbits were immunized with 100-200 μg of VLP (GII.4, GII.3 or GII.17) mixed with complete Freund's adjuvant. Three weeks later, each rabbit received a booster injection with 100-200 μg of VLP mixed with incomplete Freund's adjuvant. Two more weeks later, rabbits were bled for serum preparation. The IgG fraction of the serum was purified by rProtein A-Sepharrose Fast flow (GE Healthcare) and then dialyzed 3 times against 2 L of PBS.

2. Cultivation of intestinal epithelial cells as organoids or monolayered cells To induce intestinal epithelial cells, human iPS cell lines obtained from the University of Tokyo Institute of Medical Science / Stem Cell Bank, TkDN4-M strain (Oct3 / 4, Sox2 and Klf4 were introduced; Takayama et al., J. Exp. Med., 207: 2817-2830 2010) were used. The cells were suspended in a TC protector (DS Pharma Biomedical), stored at -80 ° C, thawed, reconstituted, and used.
Induction of intestinal epithelial cells from human iPS cells was performed according to the method described in Takahashi et al., Stem Cell Reports 10: 314-328 2018.

Organoids cultured on Matrigel (Corning) were washed with PBS and incubated at 37 ° C. for 5 minutes in TrypLE Express (Gibco) supplemented with ROCK inhibitor Y-27632 (10 μM, Wako). To separate Matrigel and organoids, the cell suspension was pipetted with a micropipette to break up cell clumps and then passed through a nylon mesh. The obtained cell suspension was added to a 5-fold volume of 10% calf serum / basic medium [Advanced DMEM / F12 (Gibco) in 10 mM HEPES (pH 7.3, Gibco), 2 mM Glutamax (Gibco) and 100 units / mL Penicillin plus 100 μg / mL streptmycin (Gibco) was added], and the mixture was centrifuged at 440 g for 5 minutes to collect the cells. The collected intestinal epithelial cells were cultured on ice on a 20% organoid culture medium supplemented with 10 μM Y-27632 [a medium obtained by adding the following to a basic medium. 25% WRN (mouse Wnt3a, human R-spondin1, human Noggin) CM (conditioned medium) (Takahashi et al., Stem Cell Reports 10: 314-328 2018), 1 × B-27 (Gibco), 50 ng / mL mouse EGF (Peprotech), 50 ng / mL human HGF (R & D systems), 10 μM SB202190 (Sigma) and 500 nM A83-01 (TGF-β receptor inhibitor, Tocris)]. The obtained cell suspension was dispensed into a 24-well plate and incubated at 37 ° C. for 10 minutes in a 5% CO ₂ incubator. Next, 500 μL of an organoid culture medium (+10 μM Y-27632) was added to each well. The average passage ratio was 1:16 or 3 × 10 ⁴ / well. The medium was replaced with a fresh organoid medium every 2-3 days. Passaging was performed every 5-7 days.

To infect HuNoV, intestinal epithelial cells that had been removed from the wells and suspended in 100 μL of an organoid culture medium (+10 μM Y-27632) in a 2.5% Matrigel-coated 96-well plate or Transwells (Corning3470) were used for 2 × 10 5 Seeded at a cell density of ⁴ / well. In the culture in Transwells, 600 μL of an organoid culture medium was added to the lower well. After culturing in a 5% CO ₂ incubator at 37 ° C. for 2 days, the medium was used as a differentiation medium [a medium obtained by adding the following to a basic medium. 12.5% RN CM (Takahashi et al., Stem Cell Reports 10: 314-328 2018), 1 × B-27, 50 ng / mL mouse EGF and 500 nM A83-01]. After further culturing for two days, the medium was replaced with a differentiation medium with or without 0.03% porcine bile (Sigma). Thereafter, the cells cultured for 2 days were used for HuNoV infection.

3. Preparation of HuNoV and Infection of Intestinal Epithelial Cells Stool of a patient infected with HuNoV was suspended in PBS to 10% (w / v). The suspension was centrifuged at 12,000 g for 30 minutes, and the obtained supernatant was passed through a 0.45 μm filter and a 0.22 μm filter. The filtered sample was aliquoted and stored at -80 ° C. Immediately before performing the infection experiments, the virus solution was diluted in basic medium to 2 × 10 ⁷ genome copies per mL. The prepared intestinal epithelial cells were infected with 100 μL thereof (2 × 10 ⁶ genomic copies) and incubated in a 5% CO ₂ incubator for 3 hours. After removing the added virus solution, the cells were washed twice with 150 μL of the basal medium. A differentiation medium containing or not containing 0.03% bile was added to 100 μL cells, and pipetted twice, and then collected as a control sample. In addition, 100 μL of differentiation medium with or without 0.03% bile was added to each well and then cultured in 5% CO ₂ for 72 hours.
For inhibition experiments, dilute the virus solution with 100 ng of anti-VLP (GII.4, GII.3, or GII.17) antibody or non-immune rabbit IgG (control IgG) before infecting cells. And incubated at 37 ° C. for 90 minutes.

4. Quantification of viral genome copies RNA was prepared from diluted virus solutions and samples collected at 3 and 72 hours post-infection using the PureLink Viral RNA / DNA Mini Kit (Invitrogen). RT-qPCR was performed using qPCR (GI / GII) Typing Kit (TakaRa) and LightCycler 480 System (Roche).

Result 1. GII.4 Antibody prepared using VLP as an antigen Compared with VLP used as an antigen, VP1 of the virus strain (17-231) contained a single amino acid mutation (D235E) in the P1 domain. Incubation of .4 HuNoV with anti-GII.4 VLP antibody completely inhibited its replication in intestinal epithelial cells (FIG. 1A).
Next, it was examined whether this anti-GII.4 VLP polyclonal antibody inhibits the replication of viruses of other GII genotypes. As a result, the anti-GII.4 VLP polyclonal antibody prepared here was unable to inhibit the replication of GII.3, GII.6 and GII.17 HuNoV (FIG. 1B). This result suggests that GII.4 VLP cannot be used as an antigen to induce neutralizing antibodies that inhibit the replication of other genotypes of HuNoV.

Incubation of GII.3 HuNoV with an anti-GII.3 VLP antibody completely inhibited its replication in intestinal epithelial cells (FIG. 2A).
Next, it was examined whether this anti-GII.3 VLP polyclonal antibody inhibits the replication of viruses of other genotypes of GII. As a result, the anti-GII.3 VLP polyclonal antibody produced here was unable to inhibit the replication of GII.4, GII.17 and GII.6 HuNoV (FIG. 2B). This result suggests that GII.3 VLP cannot be used as an antigen to induce a neutralizing antibody that inhibits replication of other genotypes of HuNoV.

2. Antibody prepared using GII.17 VLP as an antigen Pretreatment of GII.17 HuNoV with an anti-GII.17 VLP antibody inhibited the replication of GII.17 HuNoV in intestinal epithelial cells (FIG. 3A).
Next, when the effect of the anti-GII.17 VLP antibody on the replication of HuNoV of a genotype other than GII.17 was examined, the anti-GII.17 VLP antibody also inhibited the replication of GII.4 HuNoV (FIG. 3B).

The above results suggest that GII.17 VLP is highly likely to inhibit the infection of HuNoV of other genotypes classified as GII and has the ability as a multivalent vaccine antigen.

The present invention provides an antigen suitable for the purpose of preparing an antibody that inhibits HuNoV infection of a plurality of different genotypes, and the antibody. Therefore, the present invention is expected to be used in the medical field.

Claims (10)

1. A method for producing an antibody that inhibits infection of intestinal epithelial cells with a plurality of different genotypes of human norovirus (HuNoV), comprising immunizing the full length or a part of VP1 protein of GII.17.HuNoV as an antigen. A method for producing an antibody.
2. The method for producing an antibody according to claim 1, wherein the full length of the VP1 protein of {GII.17} HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
3. (3) The method for producing an antibody according to (1) or (2), wherein the plurality of different genotypes are GII.4 and GII.17.
4. {GII.17} An antibody that binds to the VP1 protein of HuNoV, which inhibits infection of multiple different genotypes of HuNoV into intestinal epithelial cells, or a functional fragment thereof.
5. The antibody or the functional fragment thereof according to claim 4, wherein the full length of the VP1 protein of {GII.17} HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
6. (6) The antibody or the functional fragment thereof according to (4) or (5), wherein the plurality of different genotypes are GII.4 and GII.17.
7. [4] A pharmaceutical composition comprising the antibody or the functional fragment thereof according to any one of claims 4 to 6.
8. {GII.17} A vaccine for suppressing infection of multiple different genotypes of HuNoV, which contains the full length or a part of the VP1 protein of HuNoV as an antigen.
9. The vaccine according to claim 8, wherein the full length of the VP1 protein of {GII.17} HuNoV comprises the amino acid sequence represented by SEQ ID NO: 3.
10. ワ ク チ ン The vaccine according to claim 8 or 9, wherein the plurality of different genotypes are GII.4 and GII.17.

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