Anti-HAV antibody

Abstract

Provided is an anti-HAV antibody usable as a prophylactic agent and a therapeutic agent for hepatitis A.　The present invention pertains to: an anti-HAV antibody or an antigen-binding fragment thereof; a pharmaceutical composition and a reagent composition containing the antibody or the antigen-binding fragment thereof; a nucleic acid encoding the antibody or the antigen-binding fragment thereof; a vector including the nucleic acid; a host cell transformed by using the nucleic acid or the vector; and a method for producing the antibody or the antigen-binding fragment thereof.

Description

Anti-HAV antibody

[0001] The present invention relates to anti-HAV antibodies.

[0002] Approximately 200-400 cases of hepatitis A are reported annually in Japan. While a vaccine is available to prevent infection, it is optional and currently most Japanese people have not been vaccinated, meaning that many do not possess protective antibodies. Hepatitis A virus can cause widespread, disseminated outbreaks via food contaminated with the virus. In 2019, there was an outbreak in Japan affecting 1,000 people, including some who died from fulminant hepatitis.

[0003] Currently, there are no antiviral drugs or other treatments, and once the disease becomes severe or fulminant, there is nothing that can be done. According to a 2016 report by the WHO, 7,000 people died from hepatitis A annually, with a mortality rate of 0.5% (Non-Patent Document 1).

[0004] Ingestion of food or drink contaminated with hepatitis A virus can lead to acute hepatitis within several weeks of the virus's entry into the body (incubation period: several weeks). In such cases, if information about contaminated food or drink is available, pre-symptomatic preventative measures may be possible. The WHO has clearly stated that early prevention, within two weeks of ingesting contaminated food or drink, can be achieved by administering a hepatitis A vaccine to induce immunity or by administering a high-titer gamma globulin preparation against hepatitis A virus (Non-Patent Document 2). While this vaccine is effective for pre-symptomatic prevention in individuals with a healthy immune system, it is not effective in individuals with a weakened immune system. Furthermore, high-titer gamma globulin preparations against hepatitis A virus can only be produced if the donor possesses neutralizing antibodies against hepatitis A virus. In most developed countries, including Japan, most blood donors lack neutralizing antibodies against hepatitis A virus, making high-titer gamma globulin preparations against hepatitis A virus difficult to obtain in Japan. In general, blood products are known to carry various risks derived from the blood donor.

[0005] Although there are treatments for fulminant hepatitis A, such as liver support therapy and liver transplantation, there are no approved drugs equivalent to antivirals that can suppress viral replication.

[0006] WHO. Hepatitis A,<https: / / www.who.int / news-room / fact-sheets / detail / hepatitis-a> (2022).https: / / www.who.int / publications / i / item / who-wer8728-29-261-276

[0007] An object of the present invention is to provide an anti-hepatitis A virus (HAV) antibody that can be used as a preventive or therapeutic agent for hepatitis A.

[0008] The present inventors first isolated IgH and IgL variable region RT-PCR fragments from peripheral blood B lymphocytes of hepatitis A vaccine recipients and cloned them into expression vectors for human IgG1-IgH (heavy chain polypeptide) and human IgG-IgLkappa (light chain polypeptide), respectively. These vectors were then transfected into HEK293 cells, and antibodies (recombinant human IgG1) secreted into the culture supernatant were obtained. Using the supernatant, HAV-specific IgG clones were screened, and 10 HAV-specific IgG clones were identified. Three of these clones exhibited infection-inhibitory activity in cultured cells, and one clone was confirmed to inhibit infection and prevent hepatitis in animal experiments. The present invention was completed based on these findings.

[0009] The gist of the present invention is as follows: (1) An antibody or an antigen-binding fragment thereof, which is any one of (a), (b), and (c) below: (a) an antibody comprising a heavy chain having a heavy-chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 1, CDRH2 comprises the amino acid sequence of SEQ ID NO: 3, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, and a light chain having a light-chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, CDRL2 comprises the amino acid sequence of DAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 15, said antibody being capable of specifically binding to hepatitis A virus (HAV); (b) an antibody comprising a heavy chain having a heavy-chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 41, CDRH2 comprises the amino acid sequence of SEQ ID NO: 43, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 45, and a light chain having a light-chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 53, CDRL2 comprises the amino acid sequence of FAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 55, said antibody being capable of specifically binding to hepatitis A virus (HAV); (c) an antibody comprising a heavy chain having a heavy-chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 41, CDRH2 comprises the amino acid sequence of SEQ ID NO: 43, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 45, and a light chain having a light-chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 53, CDRL2 comprises the amino acid sequence of FAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 55, said antibody being capable of specifically binding to hepatitis A virus (HAV); (2) An antibody or antigen-binding fragment thereof according to (1), wherein the amino acid sequences of CDRH1 to CDRL1 are determined by homology analysis using IgBlast with the nucleic acid sequences of the full-length heavy chain and full-length light chain, respectively. (3) The antibody or antigen-binding fragment thereof according to (1) or (2), characterized in that it binds to Hepatitis A virus. (4) The antibody or antigen-binding fragment thereof according to (1) or (2), wherein the heavy chain variable region comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 7, 23, 47, or 67. (5) The antibody or antigen-binding fragment thereof described in (1) or (2), wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17, 29, 57 or 75.(6) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to (1) or (2). (7) The pharmaceutical composition according to (6) for preventing hepatitis A virus infection. (8) The pharmaceutical composition according to (6) for treating hepatitis A virus infection. (9) A reagent composition comprising the antibody or antigen-binding fragment thereof according to (1) or (2). (10) A nucleic acid encoding the antibody or antigen-binding fragment thereof according to (1) or (2). (11) A vector comprising the nucleic acid according to (10). (12) A host cell transformed with the nucleic acid according to (10) or the vector according to (11). (13) A method for producing an antibody or antigen-binding fragment thereof, comprising culturing the cell according to (12) and collecting from the culture an antibody or antigen-binding fragment thereof capable of specifically binding to hepatitis A virus (HAV) from the culture.

[0010] The present invention provides an anti-HAV antibody that can be used as a preventive or therapeutic agent for hepatitis A. This specification includes the contents as disclosed in the specification and / or drawings of Japanese Patent Application No. 2024-151994, from which the present application claims priority.

[0011] Generation of human HAV-specific monoclonal antibodies. (A) Schematic diagram of the experimental workflow for generating HAV-specific monoclonal antibodies. (B) Flow cytometer analytical sorting strategy for separating plasmablasts by flow cytometry. (C) ELISA screening of HAV antigen-specific recombinant human IgG1 monoclonal antibodies using the supernatant of HEK293 cells transfected with A. Generation of human anti-HAV antibodies from plasmablasts induced with an HAV vaccine. (A and B) Screening of human anti-HAV antibodies using recombinant HAV (HM175 / 18f-NLuc) harboring a nanoluciferase (NLuc) reporter gene. Huh7.5.1 cells were infected with 100 viral genome copies per cell of enveloped HAV (eHAV) (Figure 2A) or naked HAV (nHAV) (Figure 2B) at 10 μg / ml and reacted with the respective antibodies. Intracellular NLuc activity was measured 72 hours postinfection. Anti-HAV rabbit serum from rabbits immunized with HAV antigen was used as a positive control. The vertical axis represents the NLuc value of PBS-treated infected cells, set to 1 (n = 3). (C) After infection with HAV at 100 virus copies per cell, Ab8 was reacted with nHAV at the indicated concentrations. NLuc activity in Huh7.5.1 cells was measured at 72 h postinfection. The NLuc value of PBS-treated infected cells was set to 1 (n = 3). (D) After infection of Huh7.5.1 cells, two concentrations of Ab8 (100 or 1000 ng / ml) were reacted with the indicated amounts of nHAV. The NLuc activity in the cells was measured at 72 h postinfection. The NLuc value of PBS-treated cells infected with nHAV at 300 virus copies per cell was set to 1 (n = 3). (E and F) Infection inhibition was also assessed by immunofluorescence staining. Huh7.5.1 cells were infected with 100 copies of nHAV per cell in the presence of either Ab8 (1000 ng / ml) or control hIgG. dsRNA production (Fig. 2E) and HAV 2C production (Fig. 2F) were visualized at 48 and 96 hours postinfection using the respective specific antibodies. DAPI, nuclear stain. Ab8 also inhibited two other HAV strains, KRM031 (IA) and TKM005 (IB).Huh7.5.1 cells were cultured with the indicated concentrations of Ab8 and naked HAV (KRM031 or TKM005) at 100 virus copies per cell. 72 hours postinfection, intracellular viral RNA was measured by RT-qPCR. PBS-treated controls were assigned a value of 1 (n = 3). Ab8 inhibits the viral entry step. (A) Schematic of time-of-addition experiments. (B) In pretreatment assays, Huh7.5.1 cells were cultured with Ab8 (50 or 100 ng / ml) for 2 hours, washed, and infected with nHAV / 18f-NLuc at 100 copies per cell. In pretreatment and cotreatment assays, Huh7.5.1 cells were cultured with Ab8 for 2 hours, followed by 2 hours of incubation with both HAV / NLuc and Ab8. In the post-treatment assay, Huh7.5.1 cells were infected with HAV / 18f-NLuc for 4 hours, then the virus was washed away, and fresh medium containing Ab8 was added immediately or 4 hours after washing. At 72 hours post-infection, cells were harvested and NLuc activity was measured. PBS-treated controls were assigned a value of 1 (n = 3). (C) Schematic of the HAV attachment and entry process. (A) Attachment (viral attachment) assay. The indicated concentrations of Ab8 were incubated with naked (n) HAV (HM175) at 5 x 107 / ml in 0.5 ml of PBS overnight at either 4°C or 37°C, and the mixture was incubated on ice for 30 minutes. The ice-cold mixture of nHAV and antibody was then added to pre-chilled Huh7.5.1 cells. The cells were then incubated on ice for 2 hours (200 viral genome copies per cell was used). The level of viral RNA attached to the cell surface was quantified by RT-qPCR. (B) Comparison of adhesion inhibition by Ab8 with that by other antibodies. (C) Huh7.5.1 cells were cultured on ice for 1 hour with 100 copies of naked (n)HAV / 18f-NLuc per cell in the presence of either Ab8 or gangliosides at the indicated concentrations. After a further 16 hours at 37°C, the cells were harvested and their NLuc activity was measured. The NLuc activity of the control PBS was set to 1. Comparison of Ab8 infection-inhibitory activity in humans and mice.(A and B) Huh7.5.1 cells were infected with nHAV / 18f-NLuc at 100 copies per cell (A) or 10,000 copies per cell in the presence of Ab8 antibody at the indicated concentrations. The NLuc activity of the cells was measured at 72 hours postinfection. The NLuc activity of the PBS-treated negative control was set to 1 (n = 3). Isolation and characterization of Ab8-resistant viruses. (A) Experimental procedure for isolating Ab8-resistant viruses. (B) Huh7.5.1 cells were cultured with 100 copies per cell of naked (n)HAV (either the parental virus strain (HM175) or the V162E virus strain) and the indicated concentrations of Ab8. At 72 hours postinfection, intracellular viral RNA was quantified by RT-qPCR. The RNA amount of the PBS-treated control was set to 1. (C) Comparison of viral entry efficiency between the parental and V162E virus strains. Huh7.5.1 cells were infected with nHAV (either parental HM175 or V162E) and incubated on ice for 2 hours or at 37°C for 16 hours. The amount of viral RNA attached to the cell surface (left) or intracellular viral RNA (right) was quantified by RT-qPCR. (D) Experimental design for evaluating Ab8 function by immunoprecipitation (top). HAV (parental HM175 or V162E) was incubated with Ab8 antibody and protein G beads, followed by immunoprecipitation. Viral RNA was extracted from both the flow-through fraction (FT) and the immunoprecipitation fraction (IP) and quantified by RT-qPCR. The number of copies of the viral RNA genome used in the IP reaction is defined as 100%, and the recovery rate of input virus is shown. Evolutionary conservation of V(valine)162 in the VP1 gene. The amino acid site of the V162E mutation in the VP1 gene of Ab8-resistant viruses has been shown to be conserved across six different HAV genotypes. (A) 100 μl of Aimgen (0.1 μg / ml) was plated on an ELISA plate overnight at 37°C. After blocking with 2% BSA for 2 hours at 37°C, Ab8 (human IgG1, 1 μg / ml) along with Ab8-mIgG2c (0.25-4 μg / ml) was added to the plate, and the plate was incubated for 2 hours at 37°C. Binding of Ab8 antibody to HAV antigen was measured with anti-mouse IgG HRP.The absorbance without human IgG was set to 1. The 0.5 OD level is indicated by the dotted line. (B) 100 μl of Aimgen (0.1 μg / ml) was plated onto an ELISA plate and incubated overnight at 37°C. After blocking with 2% BSA, Ab8-mIgG2c (0.25–4 μg / ml) containing Ab8 was added to the plate, and the plate was incubated at 37°C for 2 hours. Binding of Ab8-mIgG2c to HAV was measured using anti-mouse IgG HRP. The absorbance without competing antibody was set to 1. The 0.5 OD level is indicated by the dotted line. Ab8 antibody inhibited infection in vivo and prevented the development of acute hepatitis. (A) Experimental protocol for evaluating the inhibitory effect of Ab8 in a mouse model. Ifnar1 - / - deficient mice were inoculated intravenously with 10^4 genome equivalents (GE) of HAV (HM175) and, 1 day later, received intraperitoneal injections of PBS, Ab8, mIgG1-Ab8, or mIgG2c-Ab8. Feces and serum were collected at the indicated timelines. (B-E) Fecal HAV RNA (B), serum HAV RNA (C), serum ALT (D), and liver HAV RNA (E) from Ifnar1 - / - deficient mice. Data are means ± SEM; n = 4. *P < 0.05, ***P < 0.001. Pathological analysis of acute hepatitis. (A) Schematic diagram of the experimental design. Ifnar1 - / - deficient mice were inoculated intravenously with 10^4 genome equivalents (GE) of HAV (HM175) and, 1 day later, received intraperitoneal injections of PBS or Ab8-mIgG2c. Feces, serum, and liver samples were examined on days 7 or 14 postinfection. (B) Serum ALT levels and fecal and liver viral RNA levels were measured in B. (C) Representative hematoxylin and eosin (H&E)-stained livers from control (no-infection) and infected Ifnar1 - / - mice administered Ab8-mIgG2c (or PBS) at 14 dpi. Scale bar: 50 mm. Whole-slide image analysis results from the top row and bottom rows. Tissues were classified into three segments: hepatocytes (pink), blood vessels (mauve), and inflammation (green). (D) Quantification of inflammation by whole-slide image analysis in C. Results are from 15 fields per mouse.(E) Top panel: Representative anti-Iba-1 antibody-stained livers from control (no-infection) and infected Ifnar1 - / - mice administered Ab8-mIgG2c (or PBS) at 14 dpi. Nuclear staining was performed with hematoxylin. Scale bar: 50 mm. Bottom panel: Results of whole-slide image analysis of the top panel. Three tissue segments were identified: hepatocytes (mauve), blood vessels (pink), and Iba-1+ cell inflammation (brown), as well as two cell segments: Iba-1+ cells (red dots) and other cells (green dots). (F) Quantification of inflammation by whole-slide image analysis in E. Results are from 15 fields per mouse.

[0012] The present invention is described in detail below. The present invention provides any one of the following (a), (b), and (c) antibodies or antigen-binding fragments thereof: (a) an antibody comprising a heavy chain having a heavy-chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 1, CDRH2 comprises the amino acid sequence of SEQ ID NO: 3, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, and a light chain having a light-chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, CDRL2 comprises the amino acid sequence of DAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 15, said antibody being capable of specifically binding to hepatitis A virus (HAV); (b) an antibody comprising a heavy chain having a heavy-chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 41, CDRH2 comprises the amino acid sequence of SEQ ID NO: 43, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 45, and a light chain having a light-chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 53, CDRL2 comprises the amino acid sequence of FAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 55, said antibody being capable of specifically binding to hepatitis A virus (HAV); An antibody capable of specifically binding to hepatitis A virus (HAV), comprising: a heavy chain having a heavy chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 61, CDRH2 comprises the amino acid sequence of SEQ ID NO: 63, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 65; and a light chain having a light chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 71, CDRL2 comprises the amino acid sequence of GAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 73. The amino acid sequences of CDRH1 to CDRL1 to CDRL3 may be determined by homology analysis using IgBlast with the nucleic acid sequences of the full-length heavy chain and full-length light chain, respectively. The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 1, 3, and 5 are set forth in SEQ ID NOs: 2, 4, and 6, respectively.

[0013] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 13 and 15 are shown in SEQ ID NOs: 14 and 16, respectively.

[0014] The amino acid sequence DAS is encoded by the nucleotide sequence GATGCATCC.

[0015] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 41, 43 and 45 are shown in SEQ ID NOs: 42, 44 and 46, respectively.

[0016] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 53 and 55 are shown in SEQ ID NOs: 54 and 56, respectively.

[0017] The amino acid sequence FAS is encoded by the nucleotide sequence TTTGCATCC.

[0018] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 61, 63 and 65 are shown in SEQ ID NOs: 62, 64 and 66, respectively.

[0019] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 71 and 73 are shown in SEQ ID NOs: 72 and 74, respectively.

[0020] The amino acid sequence GAS is encoded by the nucleotide sequence GGTGCATCT.

[0021] The antibody or antigen-binding fragment thereof of the present invention is preferably capable of neutralizing (or inhibiting infection with) hepatitis A virus.

[0022] In the antibody of the present invention, the heavy chain variable region may comprise an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 7, 23, 47, or 67.

[0023] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 7, 23, 47 and 67 are shown in SEQ ID NOs: 8, 24, 48 and 68, respectively.

[0024] In the antibodies of the present invention, when the heavy chain variable region is not 100% identical to the amino acid sequence of SEQ ID NO: 7, 23, 47, or 67, the heavy chain variable region may have an amino acid sequence in which 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is deleted, substituted, or added in a region other than CDRH1 to CDRH3 in the amino acid sequence of SEQ ID NO: 7, 23, 47, or 67 (i.e., the framework region).

[0025] In the antibody of the present invention, the light chain variable region may comprise an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 17, 29, 57, or 75.

[0026] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 17, 29, 57 and 75 are shown in SEQ ID NOs: 18, 30, 58 and 76, respectively.

[0027] In the antibodies of the present invention, when the light chain variable region is not 100% identical to the amino acid sequence of SEQ ID NO: 17, 29, 57, or 75, the light chain variable region may have an amino acid sequence in which 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is deleted, substituted, or added in a region other than CDRL1 to CDRL3 in the amino acid sequence of SEQ ID NO: 17, 29, 57, or 75 (i.e., the framework region).

[0028] The identity of two sequences can be determined using BLAST.

[0029] In the antibodies of the present invention, the heavy chain constant region may be derived from a mammal (e.g., human, mouse, or rat), and examples include heavy chain constant regions such as those of human IgG1 (SEQ ID NOs: 9 and 49), human IgG2 (NCBI Gene ID: 3501), human IgG3 (NCBI Gene ID: 3502), human IgG4 (NCBI Gene ID: 3504), mouse IgG1 (SEQ ID NO: 25), mouse IgG2c (SEQ ID NO: 35), and rat IgG1 (NCBI Gene ID: 299354).

[0030] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 9, 49, 25 and 35 are shown in SEQ ID NOs: 10, 50, 26 and 36, respectively.

[0031] The above amino acid sequence of the heavy chain constant region may have 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid deleted, substituted, or added.

[0032] In the antibodies of the present invention, the light chain constant region may be a heavy chain constant region derived from a mammal (e.g., human, mouse, or rat), and examples thereof include light chain constant regions such as human κ chain (SEQ ID NO: 19), human λ chain (NCBI Gene ID: 3538), mouse κ chain (SEQ ID NO: 31), mouse λ chain (NCBI Gene ID: 111519), and rat λ chain (NCBI Gene ID: 100361706).

[0033] The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 19 and 31 are shown in SEQ ID NOs: 20 and 32, respectively.

[0034] The above amino acid sequence of the light chain constant region may have 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid deleted, substituted, or added.

[0035] Embodiments of the present invention include the following antibodies: - An antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 21 (for example, the human Ab8 antibody described below) - An antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 27 and a light chain comprising the amino acid sequence of SEQ ID NO: 33 (for example, the mouse Ab8-IgG1 antibody described below) - An antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and a light chain comprising the amino acid sequence of SEQ ID NO: 39 (for example, the mouse Ab8-IgG2c antibody described below) - An antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO: 59 (for example, the human Ab4 antibody described below) - An antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 69 and a light chain comprising the amino acid sequence of SEQ ID NO: 77 (for example, the human Ab10 antibody described below) The nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 11, 21, 27, 33, 37, 39, 51, 59, 69 and 77 are shown in SEQ ID NOs: 12, 22, 28, 34, 38, 40, 52, 60, 70 and 78, respectively.

[0036] The antibody of the present invention may be a monoclonal antibody, but may also be a polyclonal antibody. The antibody of the present invention may also be a human antibody, or an antibody in which a portion of the heavy and / or light chain is derived from a human and the remaining portion of the heavy and / or light chain is derived from a non-human animal (chimeric antibody).

[0037] In the present invention, an antigen-binding fragment of an antibody means a part of a full-length antibody that retains the site that specifically binds to an antigen to which the antibody binds, and examples of such fragments include Fab, F(ab)'2, Fab', Fv, single-chain antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0038] The antibodies and antigen-binding fragments thereof of the present invention can be produced as recombinant antibodies or antigen-binding fragments thereof using genetic engineering techniques. Specifically, DNA encoding the heavy and light chain genes of the antibodies of the present invention or the antigen-binding fragments of the antibodies of the present invention is synthesized, inserted into an expression vector (e.g., a plasmid, bacteriophage, or virus), and then introduced into host cells (e.g., CHO cells, HEK cells, etc.). The host cells are then cultured, allowing recombinant antibodies or antigen-binding fragments thereof to be isolated from the culture. Insect cells and baculovirus may also be used to produce the antibodies and antigen-binding fragments of the present invention. Codon optimization is preferred when synthesizing the antibody heavy and light chain genes or DNA encoding the antigen-binding fragments. The antibody heavy chain and light chain genes may be inserted into the same expression vector or into separate expression vectors. The expression vector may optionally contain a promoter, enhancer, polyadenylation signal, replication origin, selectable marker gene, secretion signal sequence, transcription terminator, etc.

[0039] The ratio of antibody heavy chain genes to light chain genes inserted into the expression vector is preferably 1:1, but this may be changed as appropriate to improve antibody expression levels. Recombinant expression vectors can be introduced into host cells using known methods such as the calcium phosphate method, DEAE-dextran method, microinjection, lipofection, electroporation, transduction, scrape-loading, and shotgun fusion. Culturing host cells transfected with a recombinant expression vector produces recombinant antibodies or antigen-binding fragments thereof in the culture. Appropriate culture conditions (e.g., medium, culture time, culture temperature, CO2 concentration) can be determined by those skilled in the art. The produced antibodies or antigen-binding fragments thereof can be recovered using known protein separation and purification methods that utilize isoelectric point, size, solubility (e.g., in water, organic solvents), affinity for certain substances (e.g., substrates, coenzymes, etc., in the case of enzymes). The present invention also provides nucleic acids encoding the above-described anti-HAV antibodies or antigen-binding fragments thereof. The present invention also provides vectors containing nucleic acids encoding the above-described anti-HAV antibodies or antigen-binding fragments thereof. The present invention also provides host cells transformed with the above-described nucleic acids or vectors. The present invention also provides a method for producing an antibody or antigen-binding fragment thereof, comprising culturing the above-described cells and collecting from the culture an antibody or antigen-binding fragment thereof capable of specifically binding to hepatitis A virus (HAV). The antibodies and antigen-binding fragments thereof of the present invention specifically bind to hepatitis A virus (HAV) and have HAV infection-inhibiting activity, and can therefore be used to prevent or treat HAV infection. The antibodies and antigen-binding fragments thereof of the present invention can also be used as research reagents for immunostaining, immunoprecipitation, ELISA, virus neutralization tests, infection inhibition experiments, Western blotting, viral load quantification, viral protein quantification, and the like.

[0040] Therefore, the present invention also provides pharmaceutical compositions comprising the above-mentioned antibody or antigen-binding fragment thereof. The pharmaceutical compositions of the present invention can be used to prevent hepatitis A virus infection. The pharmaceutical compositions of the present invention can be used to treat hepatitis A virus infection.

[0041] The antibody or antigen-binding fragment thereof of the present invention may be dissolved in a buffer solution such as PBS, physiological saline, sterile water, or the like, and, if necessary, sterilized by filtration using a filter or the like, and then administered to a subject (human or non-human animal) by injection or infusion. This solution may also contain pharmaceutically acceptable additives (e.g., salts (e.g., sodium chloride, potassium chloride, etc.), buffers (e.g., phosphate buffer, citrate buffer, etc.), amino acids (glycine, alanine, glutamine, asparagine, arginine, lysine, etc.), sugars (e.g., D-mannitol, D-sorbitol, sucrose, trehalose, etc.), surfactants (e.g., polysorbate 80, polysorbate 20, etc.), isotonicity agents, pH adjusters, etc.). These additives may not be present in the body of a subject such as a human. The amount of these formulation additives added is preferably 0.001 to 100 times, and more preferably 0.1 to 10 times, the weight (mass) of the antibody or antigen-binding fragment thereof of the present invention. The pharmaceutical composition of the present invention may be in the form of a liquid, lyophilized formulation, or the like. The administration route may be intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal, or may be intranasal or oral.

[0042] The dosage, number of administrations, and frequency of administration of the antibody or antigen-binding fragment thereof of the present invention will vary depending on the subject's symptoms, age, body weight, administration method, and administration form. For example, approximately 0.001 to 100 mg / kg body weight, preferably 1 to 10 mg / kg body weight, per adult should be administered at least once at a frequency sufficient to achieve the desired effect. The present invention also provides reagent compositions comprising the above-described antibody or antigen-binding fragment thereof. The reagent composition may contain preservatives (e.g., sodium azide), buffers, surfactants, blocking agents (e.g., gelatin), glycerol (antifreeze), and the like. In the reagent compositions of the present invention, the above-described antibody or antigen-binding fragment thereof may be labeled with an enzyme (e.g., HRP, AP), a fluorescent dye (e.g., FITC, Alexa Fluor), biotin, a radioisotope (e.g., I-125, I-131), a metal particle (e.g., iron, gold), or the like.

[0043] The present invention will be described in detail below with reference to examples.

[0044] Results and Discussion: Figure 1 illustrates how Ab8 (and Ab4 and Ab10) were generated. First, as shown in A, single-cell sorting was performed on peripheral blood B cells (plasmablasts) from recipients of hepatitis A vaccine. Gene fragments encoding the variable regions of IgH and IgL isolated from each well by RT-PCR were cloned into expression vectors for human IgG1-IgH (heavy chain polypeptide) and human IgG-IgLkappa (light chain polypeptide), respectively. These were transfected into HEK293 cells, and the antibody (recombinant human IgG1) secreted into the culture supernatant was obtained. Using the supernatant, HAV-specific IgG1 was screened by ELISA. Aimgen (human vaccine antigen) was used as the ELISA antigen, and clones were selected based on their binding activity. As shown in Figure 2C, 10 HAV-specific IgG clones were identified by ELISA assay. As can be seen in the figure, Ab8, Ab4, and Ab10 are the three most reactive clones (see "Materials and Methods" for details).

[0045] As shown in Figure 1, human monoclonal antibodies were obtained from peripheral blood mononuclear cells (PMBCs) of volunteers who received the Aimgen vaccine. In Figure 2, the activity of inhibiting viral infection was measured using the human hepatocyte cell line Huh7.5.1.

[0046] In Figure 2, we used enveloped and non-enveloped (naked) HAV HM175 18f-NLuc viruses. This infection experiment system evaluates infection by NLuc activity. Using this system, Ab4, Ab8, and Ab10 antibodies significantly reduced infection efficiency in both enveloped and enveloped-naked HAVs (Figure 2A and B). (Note: Recent studies have reported that viruses exist in two types: naked, which are uncapped particles, and enveloped HAVs (eHAVs), which are enveloped with membrane components. Typically, environmental sources of infection (food and water containing viruses) are all naked HAVs. NHAVs (eHAVs) were not used in any experiments other than those shown in Figure 2.)

[0047] The rabbit antiserum, prepared by immunizing rabbits with HAV antigens (antiserum designated "anti-sera" in the figure), was used as a positive control to demonstrate inhibitory activity. The rabbit antiserum also reduced NLuc activity against enveloped and naked HAV (Fig. 2A and B).

[0048] Although enveloped HAV was relatively resistant to these monoclonal antibodies, Ab4, Ab8, and Ab10 were able to halve NLuc activity (Fig. 2A vs. Fig. 2B).

[0049] Among Ab1-10, Ab8 showed the strongest inhibitory activity against naked (n)HAV (Fig. 2B). In an assay in which nHAV 18f-NLuc was infected into Huh7.5.1 cells, the IC50 of Ab8 was 6.0 ng / ml, and this inhibition was confirmed even when the virus dose was increased to 300 genome copies per cell (Fig. 2C and 2D).

[0050] Immunostaining assays also demonstrated that the production of viral products (2C protein and viral RNA) caused by HAV infection was inhibited in the presence of Ab8, confirming the infection-inhibitory activity of Ab8 (Fig. 2E and 2F).

[0051] The inhibitory activity of Ab8 against HAV strains other than HAV18f was examined in Figure 3. As a result, Ab8 inhibited infection with KRM031 (genotype IA) and TKM005 (genotype IB).

[0052] Considering the results of Figure 2, the infection-inhibitory activity of Ab8 was reproduced with multiple virus strains, suggesting that Ab8 has infection-inhibitory activity against various genotypes present in nature.

[0053] To explore how Ab8 inhibits HAV infection, we performed a time-of-addition experiment. The experimental design is shown in Figure 4A. Pretreatment of Huh7.5.1 cells and posttreatment at 4 h postinfection with Ab8 did not result in any inhibition. However, pretreatment, cotreatment, and posttreatment, which allow Ab8 the opportunity to bind to HAV virus, reduced NLuc activity in Ab8-treated Huh7.5.1 cells (Figure 4B).

[0054] As shown in Fig. 1C, Ab8 was identified by screening of an ELISA assay using HAV antigen, suggesting that Ab8 directly binds to and inhibits HAV virus.

[0055] This experiment supported the idea that direct binding of Ab8 to the virus is the mechanism of inhibitory activity against Ab8.

[0056] Next, the present inventors investigated the HAV entry process in detail to further investigate the mechanism of action of Ab8.

[0057] The initial process of HAV entry is the attachment of the HAV virus to cell surface molecules expressed on hepatocytes, which is thought to occur even at 4°C (see schematic diagram at the bottom of Figure 4B).

[0058] The results in Figure 5A show that viral attachment was not inhibited at 4°C by any antibody (Ab8 or rabbit antiserum). On the other hand, when HAV was treated with Ab8 at 37°C (the body temperature), viral attachment was significantly inhibited, even at a concentration of 100 ng / ml.

[0059] Interestingly, the rabbit antiserum exhibiting infection-inhibitory activity (Fig. 2B) did not strongly inhibit virus adhesion (Fig. 5A).

[0060] Two additional mouse monoclonal antibodies (IgG1 class and IgG2c class) were generated by transferring the variable region gene segments of the Ab8 IgH / L gene into a mouse IgH / L expression vector and designated Ab8-mIgG1 and Ab8-mIgG2c.

[0061] The infection-inhibitory activities of Ab8, Ab8-mIgG1, and Ab8-mIgG2c were compared in a cell culture system (nHAV 18f-NLuc), and the results are shown in Figure 6. The results showed that both mouse Ab8 antibodies had infection-inhibitory activity comparable to that of Ab8 (human) antibody.

[0062] Furthermore, Ab8-mIgG2c showed slightly stronger activity than Ab8.

[0063] Adhesion assays were performed as described in Fig. 5A, including mouse Ab8 (Ab8-mIgG1 and Ab8-mIgG2c). The results showed that human Ab8 and two mouse Ab8 antibodies significantly reduced virus adhesion (Fig. 5B).

[0064] In Figure 5C, we investigated whether Ab8 could inhibit the HAV cell entry process up to 16 hours after the virus was introduced into the cells. Unlike the contact assays in Figures 5A and 5B, in these assays, HAV and Ab8 were added directly to the culture medium, without any prior antibody-virus reaction.

[0065] A paper published by Dr. S. Lemon of the United States (Nat Microbiol. 2020 Sep;5(9):1069-1078. DOI: 10.1038 / s41564-020-0727-8) showed that adding gangliosides to cultured cells can inhibit the entry process of HAV. Therefore, the present inventors compared the inhibition of viral entry between Ab8 and gangliosides.

[0066] As shown in Figure 5C, the viral entry process was strongly inhibited by Ab8 (25-1000 ng / ml). As previously reported by Dr. Lemon, inhibition was also confirmed by GD1a (the gangliosides GD1a, GM1, GM3, and GD3 used in this study were purchased from Funakoshi).

[0067] These results suggest that Ab8 inhibits HAV entry in two phases: the attachment process and the late entry process. The first phase inhibits virus attachment to the cell surface. The second phase inhibits the late entry process (the process in which the virus enters the cytoplasm from the endosome) (37°C-dependent).

[0068] To identify the Ab8 epitope, we attempted to isolate Ab8-resistant viruses. We infected Huh7.5.1 cells with the virus and allowed the virus to replicate, constantly adding Ab8 to the culture medium. This process was repeated for eight consecutive weeks. The same experiment was repeated twice, yielding the same results. Specifically, we identified a mutant virus with the V162E mutation in the VP1 gene (a substitution of glutamic acid for valine at position 162 in the VP1 gene) (Figure 7A).

[0069] As shown in Figure 8, the amino acid sequence of V162 and its surrounding sequences are conserved in at least three typical genotypes found in nature.

[0070] To reconfirm the resistance activity against Ab8, infection inhibition experiments were performed using the parental virus and the V162E strain.

[0071] As shown in Fig. 7B, the infection of V162E virus was not significantly inhibited even in the presence of Ab8 (3-100 ng / ml), and V162E virus replicated even in the presence of Ab8, whereas the infection of the parental virus strain was significantly inhibited by the addition of Ab8 (3-100 ng / ml), as expected.

[0072] The increased viral entry process of the V162E strain may have contributed to its Ab8 inhibition evasion activity. In fact, the viral entry process was comparable to that of the parent virus strain (Fig. 7C).

[0073] To investigate the mechanism by which the V162E mutation confers resistance to Ab8 inhibition, immunoprecipitation assays were performed.

[0074] To evaluate the immunoprecipitation process, viral RNA was quantified in the immunoprecipitated fraction (IP) and the flow-through fraction (FT).

[0075] As controls, immunoprecipitation was performed using control IgG (Ctrl-hIgG) and a mock control (control with no antibody added = beads only) and compared with the immunoprecipitation experiment using Ab8.

[0076] As shown in Figure 7D, the majority (70.9%) of the parental virus (HAV HM175) used in the IP reaction was recovered by Ab8 immunoprecipitation. In contrast, in the control IP reaction (Ctrl-hIgG and beads only) (right), most of the parental virus (86.4% and 83.9%) migrated to the FT fraction. In the case of V162E virus, most of the virus (98.2–97.9%) migrated to the FT fraction, even after IP with Ab8 antibody (right).

[0077] In summary, Ab8 bound to and captured most of the parent virus, whereas under the same conditions, the V162E virus was hardly captured by Ab8.

[0078] Ab8 directly binds to HAV and neutralizes it (or inhibits infection), and this binding requires V162 of VP1. The V162E mutation avoids Ab8 binding, minimizing Ab8's inhibitory activity by escaping Ab8 binding. V162 is a conserved region among HAV genotypes found in nature, and Ab8's activity against many HAV genotypes is due to the conservation of this binding region among various genotypes. It can be said that V162 of VP1 is essential for Ab8 binding to the virus, suggesting that V162 is the core epitope of Ab8.

[0079] As shown in Figure 9A, right, a competitive ELISA was performed to compare the binding properties of the Ab8 antibody with those of Ab8-mIgG2c.

[0080] Because a large amount of highly purified HAV virus is essential for accurate analysis by competitive ELISA, we used Eimgen as the HAV antigen. Eimgen is a formalin-fixed, inactivated virus produced from recombinant yeast and is used as a vaccine in Japan.

[0081] Aimgen was immobilized directly onto ELISA plates, and the indicated antibodies were then added to compete for binding with HAV.

[0082] Ab8 and Ab8-mIgG2c have the same amino acid sequence in the variable region but different amino acid sequences in the constant region.

[0083] Thus, Ab8 (human IgG1) and Ab8-mIgG2c compete for the same epitope.

[0084] The results in Figures 9A and 9B suggest that differences in the constant regions affect the binding activity of the antibodies, resulting in stronger binding of Ab8-mIgG2c than Ab8.

[0085] Using a mouse model of acute HAV hepatitis (Figure 10A), we investigated whether Ab8 inhibits HAV infection in vivo. Naked HAV with 10 genome copies (GE) was intravenously administered to Ifnar1- / - mice (groups of 5 mice). One day after administration, Ab8, Ab8-mIgG1, or Ab8-mIgG2c (control, PBS) was administered intraperitoneally (Figure 10A). In control PBS-treated mice, ALT (a hepatitis marker) rapidly increased on day 14 postinfection and then gradually decreased (Figure 10D). In contrast, the increase in ALT was minimal in the Ab8, Ab8-mIgG1, or Ab8-mIgG2c-treated groups. Consistent with the ALT results, viral shedding and viremia in the liver were significantly suppressed in the Ab8, Ab8-mIgG1, or Ab8-mIgG2c-treated groups compared with the PBS-treated group (Fig. 10B, C, D, E).

[0086] In another set of mice, the pathological status of HAV-infected mice was evaluated, as shown in FIG. 11A.

[0087] Since the peak of ALT increase was observed on day 14 after infection, we performed pathological analysis of livers collected from mice on 14 dpi.

[0088] The viral RNA levels in ALT and feces and liver at 7 and 14 dpi were consistent with the trends shown in Figure 10 (Figure 11B).

[0089] Pathological analysis of the livers at 14 dpi revealed mononuclear cell infiltration in infected (PBS-treated) livers, whereas mononuclear cell infiltration in Ab8-mIgG2c-treated mice was reduced to the level of uninfected mice (Fig. 11C and 11D).

[0090] Immunohistochemical analysis using the macrophage marker Iba-1, an inflammatory marker (FIGS. 11E and 11F), also yielded results similar to those in FIGS. 11C and 11D.

[0091] In a mouse model, a single intravenous administration of Ab8 prevented the onset of acute hepatitis and strongly suppressed viral replication, suggesting that even a single administration could be effective in preventing infection and suppressing post-exposure disease.

[0092] Isolation of HAV Vaccine-Induced Plasmablasts. The isolation strategy for vaccine-induced plasmablasts is shown diagrammatically in Figure 1B. Peripheral blood samples were collected one week after vaccination from volunteers who received two HAV vaccinations (Aimgen, KM Biologics Co., Ltd., Kumamoto, Japan) and agreed to participate in this study. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphocyte Separation Medium 1077 (PromoCell GmbH, Heidelberg, Germany) and stored at -135°C until use. Thawed PBMCs were suspended in R10 medium (RPMI-1640 medium supplemented with 10% fetal bovine serum [FBS], 100 μg / ml penicillin, 100 μg / ml streptomycin, and 1% GlutaMAX Supplement [Thermo Fisher Scientific, Waltham, MA, USA]) and incubated at 37°C for 2 hours in the presence of DNase I (20 μg / ml). After incubation, the cells were washed and suspended in DMEM supplemented with 2% FBS and then treated with FcR blocking reagent, human monoclonal antibodies against human CD2 (1:400, RPA-2.10, BioLegend, San Diego, CA, USA), CD4 (1:400, RPA-T4, BioLegend), IgD (1:400, IA6-2, BD Bioscience, San Diego, CA, USA), and IgM (1:100, MHM-88, BioLegend) (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany), and biotinylated monoclonal antibodies for 30 min at 4°C. Cells were negatively enriched using the MACS system with streptavidin microbeads (Miltenyi Biotec) and then treated with biotinylated monoclonal antibodies against human CD10 (1:400, eBioCB-CALLA, Thermo Fisher Scientific) and CD14 (1:400, 63D3, BioLegend) in addition to the original biotinylated monoclonal antibodies for 30 min at 4°C.After washing and suspending in B cell medium (RPMI-1640 medium supplemented with 10% FBS, 100 IU / mL penicillin, 100 μg / mL streptomycin, 55 μM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, and 1% MEM non-essential amino acids), the cells were stained with Live / Dead Fixable Aqua Dead Cell Stain (1:500, ThermoFisher Scientific), Brilliant Violet 785-conjugated anti-human CD19 antibody (1:100, HIB19, BioLegend), Pacific Blue-conjugated anti-human CD20 antibody (1:100, 2H7, BioLegend), Alexa Fluor 700-conjugated anti-human CD27 antibody (1:100, O323, BioLegend), FITC-conjugated anti-human CD38 antibody (1:100, HIT2, ThermoFisher Scientific), and Brilliant Violet 785-conjugated anti-human CD38 antibody (1:100, HIT2, ThermoFisher Scientific). Cells were stained with 510-conjugated streptavidin (1:200, BioLegend) for 30 minutes at 4°C. Plasmablasts (CD19+CD20-CD27+CD38+) were isolated as single cells into 96-well plates using a FACSAria III (BD Biosciences), excluding CD2+, CD4+, CD10+, CD14+, IgD+, IgM+, and dead cells.

[0093] Generation of Human and Mouse Monoclonal Antibodies. Recombinant human monoclonal antibodies were prepared using the method described by Tiller et al. (Tiller et al. 2008, Tiller et al. 2009, Tonouchi et al. 2020). The nucleotide sequences of RT-PCR products from VH and VL genes of single-cell sorted plasmablasts were determined (Eurofins Genomics, Tokyo, Japan), and the V(D)J genes were identified using IgBLAST (http: / / www.ncbi.nlm.nih.gov / igblast). To generate human IgG1, different pairs of VH and VL genes were selected and subcloned into the respective heavy chain expression vectors (Tiller et al. 2008) and light chain expression vectors (Tiller et al. 2008). The heavy chain and light chain expression vector pairs were transfected into HEK293 cells using a transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Supernatants were collected 4 days after transfection and screened for reactivity to HAV antigens by enzyme-linked immunosorbent assay (ELISA) as described above and next (see Fig. 1A, C).

[0094] To produce recombinant mouse monoclonal antibodies (mouse IgG1 and mouse IgG2c), the variable region sequences of the VH and VL genes of human Ab8 were amplified with the primers shown below and subcloned into expression plasmids for mouse IgG1, mouse IgG2c, and mouse Igk using the infusion reaction method (Takara Bio). The expression plasmids for mouse IgG2c and mouse Igk were used in reference paper 17, but these three expression plasmids were created by replacing the constant regions of the human heavy chain and light chain expression vectors mentioned above with those of mouse IgG1, IgG2c, and mouse Igk, respectively. The primers used for subcloning by infusion reaction were as follows: pMiceIgk ab8FW (5'-TAGCAACTGCAACCGGTGTACATTCAGAAATTG-3': SEQ ID NO: 79) and pMiceIgk ab8RV (5'-GTTGGTGCAGCATCCGTACGTTTGATCTCCAGC-3': SEQ ID NO: 80); pMiceIgG1 Ab8FW (5'-GTAGCAACTGCAACCGGTGTACATTCCCAGGTGCA-3': SEQ ID NO: 81) and pMiceIgG1 Ab8RV (5'-AGATGGGGGTGTCGTCGACGCTGAGGAGACGGTG-3': SEQ ID NO: 82); pMiceIgG2c Ab8FW (5'-GTAGCAACTGCAACCGGTGTACATTCCCAG-3': SEQ ID NO: 83) and pMiceIgG2c Ab8RV (5'-CTGGGGAGCGGTTGTCGACGCTGAGGAGACGGTG-3': SEQ ID NO: 84). After constructing the expression plasmid, the antibody was produced using the same process as for human Ab8.

[0095] Screening for HAV antigen-specific recombinant human IgG1 monoclonal antibodies (ELISA). To screen for HAV antigen-specific recombinant human IgG1 monoclonal antibodies, ELISA was performed. First, the amount of human IgG in the transfection supernatant was measured by ELISA. ELISA plates (F96 Maxisorp Nunc-Immuno plate, Thermo Fisher Scientific) were coated overnight at 4°C with anti-human IgG Fab-specific polyclonal antibody (#15260, Sigma-Aldrich, Taufkirchen, Germany) at a concentration of 1 μg / mL in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3; pH 9.6). After washing with phosphate-buffered saline (PBST) containing 0.05% Tween 20, the wells were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for 45 minutes at room temperature. After washing with PBST, 50 μL of supernatant sample diluted with Can Get Signal Solution 1 (TOYOBO, Osaka, Japan) was added to the wells and incubated for 2 hours at room temperature. After washing with PBST, 100 μL of horseradish peroxidase (HRP)-conjugated anti-human IgG (SouthernBiotech, Birmingham, AL) diluted (1:5000) in Can Get Signal Solution 2 (TOYOBO) was added to the wells and incubated for 1 hour at room temperature. After washing with PBST, HRP activity was visualized by adding TMB substrate and stopped by adding 2N sulfuric acid. Absorbance was measured at a dominant wavelength of 450 nm and a secondary wavelength of 630 nm using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA).

[0096] Supernatant samples with a human IgG concentration of 1 μg / mL were then prepared using Can Get Signal Solution 1. To test for reactivity to HAV antigens, the assay was performed as described above using ELISA plates coated overnight at 4°C with Aimgen at a concentration of 325 ng / mL in carbonate buffer (no Aimgen served as a negative control). Net absorbance was calculated by subtracting the value for uncoated wells from that for Aimgen-coated wells. The cutoff value was determined as the mean net absorbance of the blank wells + 6 SD.

[0097] The human IgG1 monoclonal antibody used as a negative control in this study was established as a human antibody against norovirus and has been published in a paper (Onodera et al. 2019, doi:10.4049 / jimmunol.1900481 (2019).) The mouse IgG monoclonal antibody used as a negative control was purchased from Sigma-Aldrich.

[0098] Huh7.5.1 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Cell Culture Bioscience), 10 units / ml penicillin, and 10 mg / ml streptomycin at 37°C and 5% CO .

[0099] Virus Preparation: HM175 / 18f-NLuc recombinant virus (HM175 / 18f-NLuc, HAV / NLuc) and HAV (HM175 / 18f) were obtained from Dr. Stanley Lemon at the University of North Carolina at Chapel Hill. HAV / NLuc assays were performed as previously described. HAV-infected cells were cultured for 10 days. The culture supernatant containing enveloped HAV (eHAV) was harvested by centrifugation at 1,000 g for 10 min at 4°C, and the cells were removed. To purify naked HAV (nHAV), the eHAV-containing supernatant was treated with NP-40 (final concentration 2%) and centrifuged twice at 10,000 g for 30 min to remove debris. The supernatant was concentrated by ultracentrifugation at 100,000 g for 2 h at 4°C, and the precipitated fraction was resuspended in PBS. Isopycnic centrifugation of the virus was performed as previously described. The precipitated fractions were resuspended in PBS, overlaid with 8–40% iodixanol (Opti-Prep), and centrifuged at 141,000 g in an SW41-Ti rotor in an Optima L-90K ultracentrifuge for 24 hours at 4°C. Approximately 20 fractions were collected from the top of the gradient, and density was calculated by weighing 100 μl of each fraction. These fractions (approximately 1.22–1.28 g cm−3) were combined and the buffer was replaced with PBS using an Amicon filter (Millipore, AMICON ULTRA-15 100 kDa cutoff).

[0100] HAV / NLuc Assay After infection with HAV / NLuc virus, intracellular NLuc was determined using the Nano-Glo Luciferase Assay System (Promega). Nanoluciferase activity was measured according to the kit protocol.

[0101] Quantification of viral genome RNA levels. Ab8 antibody or PBS was mixed with virus at 100 viral genome copies per cell and incubated. HAV strains (KRM031 and TKM005) were mixed with Ab8 antibody at different concentrations and incubated with Huh7.5.1 cells for 72 hours. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN), and HAV RNA was quantified by RT-qPCR. RNA was extracted from mouse feces and serum using the QIAamp Virus Isolation Kit (QIAGEN). RT-qPCR was performed using the THUNDERDIRD™ Probe One-step qRT-PCR Kit (TOYOBO) and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The forward primer was 5′-AGGGTAACAGCGG CGGATAT-3′ (SEQ ID NO: 85), and the reverse primer was 5′-ACAGCCCTGACARTCAATYCMCT-3′ (SEQ ID NO: 86). The TaqMan probe was FAM-5'-AGACAAAAACCATTCAACRCCGRAGGAC-3'-TAM (SEQ ID NO: 87). The one-step RT-qPCR protocol was as follows: 50°C for 5 minutes, 95°C for 30 seconds, 40 cycles of 95°C for 3 seconds, 60°C for 30 seconds.

[0102] Immunofluorescence assay (IFA). As described above, cells were fixed with 4% paraformaldehyde (Nacalai Tesque) in PBS for 1 hour and permeabilized with 0.1% Triton X-100 (Nacalai Tesque) in PBS for 30 minutes. HAV 2C protein was detected with a primary antibody (1:500 dilution; Abnova). dsRNA was detected with a primary antibody (1:200 dilution; SCICONS). Nuclei were stained with DAPI (Thermo Scientific™). Images were taken with a ZEISS LSM710 confocal microscope.

[0103] Isolation of Ab8-resistant HAV virus: Naked HAV (107 GE / mL) was reacted with 0.1 μg / mL Ab8 antibody and then infected into Huh7.5.1 cells. Culture supernatants were harvested when half of the cells had died, followed by production and isolation of naked virus. This virus treatment and incubation cycle was repeated eight times until the Ab8 antibody could no longer inhibit HAV infection. Viral genomic RNA was extracted from the parental and Ab8-resistant viruses and reverse transcribed using SuperScript III Reverse Transcriptase (RT). The viral genome sequences from VP4 to pX were determined by Sanger sequencing and compared between the parental and Ab8-resistant viruses. The V162E mutation in the VP1 gene was identified as the same mutation in two independent experiments. The viral region from VP4 to pX was cloned by PCR using the following synthetic primers: FW (5'-ATGAACATGTCTAGACAAG-3' (SEQ ID NO: 88)) and RV (5'-TTGTGAAAACAGTCCCTTC-3' (SEQ ID NO: 89))

[0104] The sequence of the PCR product was analyzed by Sanger sequencing using the following primers: Fw2 (5'-GGATTGATCTGTGCTATGG-3' (SEQ ID NO: 90)); Fw3 (5'-TGGTTGGGATCAAAATTACTC-3' (SEQ ID NO: 91)); Fw4 (5'-AGGTGAGTACACTGCCATTG-3' (SEQ ID NO: 92)); Fw5 (5'-TATGGGAAGGTCTCATTTC-3' (SEQ ID NO: 93)); Fw6 (5'-CTGTCACAGAACAATCAGAG-3' (SEQ ID NO: 94)) and RV2 (5'-TATCAACAGAGGTTCTCAAAG-3' (SEQ ID NO: 95)).

[0105] Immunoprecipitation (IP) Experiments: 50 μl of SureBeads Protein G (Bio-Rad) was washed with the immunoprecipitation (IP) reaction solution (25 mM HEPES-KOH [pH 7.9], 200 mM NaCl, 5 mM MgCl2, 0.2% NP-40, 10% glycerol, 1 mM dithiothreitol [DTT]). 1 μg / ml of Ab8, Ab8-mIgG1, Ab8-mIgG2c, R10, mouse control IgG (Sigma-Aldrich), and human control IgG (anti-human norovirus monoclonal antibodies) was mixed with the Protein G beads. Naked HAV was then added and incubated overnight at 4°C with rotation. The IP complexes were collected and washed five times with 1 ml of IP reaction solution. Viral RNA was extracted from the beads containing the IP complexes and the flow-through (FT) and measured by RT-qPCR.

[0106] HAV infection challenge of mice. Ifnar1- / - knockout (KO) mice were provided by Dr. S. Morikawa of the National Institute of Infectious Diseases. Mice were bred and housed at the National Institute of Infectious Diseases in accordance with the policies and guidelines of the Institutional Animal Care and Use Committee (IACUC). All experiments using mice were approved by the National Institute of Infectious Diseases IACUC.

[0107] HM175 virus is infectious and amplified in Ifnar1- / - KO mice, and in this study, the 12th passage was used to prepare liver inocula. Livers were homogenized in PBS and centrifuged at 10,000 × g for 30 minutes. As previously described, the supernatant was aliquoted and stored at -80°C. Liver HAV RNA (GE) levels were quantified by real-time RT-qPCR. Ifnar1- / - KO mice were infected at 6–10 weeks of age by intravenous inoculation with 10 genome equivalents (GE) of HAV. The day after inoculation, 500 μg of each antibody, including Ab8, mIgG1-Ab8, or mIgG2c-Ab8, was intraperitoneally inoculated with PBS. Infected mice were maintained, feces were collected, and serum samples were periodically collected. Tissues were collected at necropsy and preserved in RNAlater (Thermo Fisher Scientific, Waltham, MA) or fixed in 10% neutral phosphate-buffered formalin for 48 hours and then stored in 70% ethanol until histological processing. RNA was extracted from serum and fecal samples using the QIAamp viral RNA isolation kit. Liver HAV RNA was isolated from tissues using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. RNA concentration was measured using a NanoDrop 2000c (Thermo Fisher Scientific). HAV RNA was quantified by real-time RT-qPCR (TOYOBO). Serum ALT was measured using a DRI-CHEM NX700 (FUJIFILM) and the GPT / ALT-PIII kit. Mouse livers were treated with 10% neutral formalin buffer (Wako) for 24 hours and then replaced with 70% ethanol.

[0108] Materials and Methods for Mouse Pathological Analysis: To obtain animal tissues, mice were anesthetized, and liver tissue was harvested and fixed in 10% phosphate-buffered formalin. Fixed tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). For immunohistochemical staining, antigen retrieval of formalin-fixed mouse tissue sections was performed by autoclaving at 121°C for 10 minutes in a pH 6.0 retrieval solution (Nichirei, Tokyo, Japan). To detect macrophages, rabbit anti-human Iba-1 antibody (019-19741; Wako Pure Chemical Industries, Osaka, Japan) was used. Diaminobenzidine (Sigma-Aldrich) was used as a color developer to visualize the enzymatic activity of HRP. Nuclei were stained with hematoxylin for 10 seconds.

[0109] Tissue phenotyping was performed using whole-slide imaging analysis (PhenoImager Fusion, AKOYA Biosciences) followed by inForm tissue analysis software (AKOYA Biosciences).

[0110] Research Ethics: The use of human blood was approved by the National Institute of Infectious Diseases Human Subjects Ethics Review Committee (Permit Number: 913). Participants provided written informed consent in accordance with the Declaration of Helsinki before enrollment.

[0111] Statistical analysis was performed using GraphPad Prism 8. Data are presented as mean ± SEM. Comparisons between multiple groups were analyzed by one-way ANOVA or t-test. A P value of less than 0.05 was considered statistically significant.

[0112] Reference papers 1 WHO. Hepatitis A, <https: / / www.who.int / news-room / fact-sheets / detail / hepatitis-a> (2022). 2 Kiyohara, T. et al. Seroepidemiology of hepatitis A virus infection in Japan: An area of very low endemicity. Microbiol Immunol 67, 14-21, doi:10.1111 / 1348-0421.13035 (2023). 3 Lu, P. J. et al. Surveillance of Vaccination Coverage Among Adult Populations -United States, 2018. MMWR Surveill Summ 70, 1-26, doi:10.15585 / mmwr.ss7003a1 (2021). 4 Victor, J. C. et al. Hepatitis A vaccine versus immune globulin for postexposure prophylaxis. N Engl J Med 357, 1685-1694, doi:10.1056 / NEJMoa070546 (2007). 5 Zhong, J. et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 102, 9294-9299, doi:10.1073 / pnas.0503596102 (2005). 6 Shi, S. et al. Novel flavonoid hybrids as potent antiviral agents against hepatitis A: Design, synthesis and biological evaluation. Eur J Med Chem 238, 114452, doi:10.1016 / j.ejmech.2022.114452 (2022). 7 Yamane, D.et al. Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses. Nat Microbiol 4, 1096-1104, doi:10.1038 / s41564-019-0425-6 (2019). 8 Feng, Z. et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 496, 367-371, doi:10.1038 / nature12029 (2013). 9 Yoneyama, T. et al. Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay. J Virol Methods 145, 162-168, doi:10.1016 / j.jviromet.2007.05.023 (2007). 10 Zheng, X., Ohsaki, E. & Ueda, K. Mechanism of angiopoietin-1 upregulation in Kaposi's sarcoma-associated herpesvirus-infected PEL cell lines. J Virol 89, 4786-4797, doi:10.1128 / JVI.03144-14 (2015). 11 Onodera, T. et al. Immune-Focusing Properties of Virus-like Particles Improve Protective IgA Responses. J Immunol 203, 3282-3292, doi:10.4049 / jimmunol.1900481 (2019). 12 Tani, H. et al.Efficacy of T-705 (Favipiravir) in the Treatment of Infections with Lethal Severe Fever with Thrombocytopenia Syndrome Virus. mSphere 1, doi:10.1128 / mSphere.00061-15 (2016). 13 Shiota, T. et al. Macrophage Depletion Reactivates Fecal Virus Shedding following Resolution of Acute Hepatitis A in Ifnar1(- / -) Mice. J Virol 96, e0149622, doi:10.1128 / jvi.01496-22 (2022). 14 Fricks, C. E. & Hogle, J. M. Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding. J Virol 64, 1934-1945, doi:10.1128 / JVI.64.5.1934-1945.1990 (1990). 15 Lemon, S. M., Jansen, R. W. & Newbold, J. E. Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl. J Virol 54, 78-85, doi:10.1128 / JVI.54.1.78-85.1985 (1985). 16 Siegl, G. & Frosner, G. G. Characterization and classification of virus particles associated with hepatitis A. I.Size, density, and sedimentation. J Virol 26, 40-47, doi:10.1128 / JVI.26.1.40-47.1978 (1978). 17 Tonouchi, K. et al. Stereotyped B-cell response that counteracts antigenic variation of influenza viruses. Int Immunol 32, 613-621, doi:10.1093 / intimm / dxaa038 (2020). 18 Tiller, T., Busse, CE & Wardemann, H. Cloning and expression of murine Ig genes from single B cells. J Immunol Methods 350, 183-193, doi:10.1016 / j.jim.2009.08.009 (2009). 19 Tiller, T. et al. Efficient generation of Monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329, 112-124, doi:10.1016 / j.jim.2007.09.017 (2008). All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0113] The anti-HAV antibody of the present invention can be used as a preventive agent for infection with hepatitis A virus or a therapeutic agent for hepatitis A. It can also be used as a reagent.

[0114]

Claims

1. An antibody or antigen-binding fragment thereof selected from the group consisting of (a), (b), and (c) below: (a) an antibody comprising a heavy chain having a heavy chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 1, CDRH2 comprises the amino acid sequence of SEQ ID NO: 3, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 5, and a light chain having a light chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 13, CDRL2 comprises the amino acid sequence of DAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 15, said antibody being capable of specifically binding to hepatitis A virus (HAV); (b) an antibody comprising a heavy chain having a heavy chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 41, CDRH2 comprises the amino acid sequence of SEQ ID NO: 43, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 45, and a light chain having a light chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 53, CDRL2 comprises the amino acid sequence of FAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 55, said antibody being capable of specifically binding to hepatitis A virus (HAV); an antibody comprising: a heavy chain having a heavy chain variable region in which CDRH1 comprises the amino acid sequence of SEQ ID NO: 61, CDRH2 comprises the amino acid sequence of SEQ ID NO: 63, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 65; and a light chain having a light chain variable region in which CDRL1 comprises the amino acid sequence of SEQ ID NO: 71, CDRL2 comprises the amino acid sequence of GAS, and CDRL3 comprises the amino acid sequence of SEQ ID NO: 73, wherein the antibody is capable of specifically binding to hepatitis A virus (HAV).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequences of CDRH1 to CDRH3 and CDRL1 to CDRL3 are determined by homology analysis using IgBlast with the nucleic acid sequences of the full-length heavy chain and full-length light chain, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which binds to hepatitis A virus.

4. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, 23, 47 or 67.

5. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17, 29, 57, or 75.

6. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. The pharmaceutical composition according to claim 6 for preventing hepatitis A virus infection.

8. The pharmaceutical composition according to claim 6 for treating hepatitis A virus infection.

9. A reagent composition comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

10. A nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1 or 2.

11. A vector comprising the nucleic acid of claim 10.

12. A host cell transformed with the nucleic acid of claim 10 or the vector of claim 11.

13. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the cell of claim 12 and recovering from the culture an antibody or an antigen-binding fragment thereof capable of specifically binding to hepatitis A virus (HAV).

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