CN119119256A

CN119119256A - Monoclonal antibody against Japanese encephalitis virus and Zika virus and its application

Info

Publication number: CN119119256A
Application number: CN202411363873.9A
Authority: CN
Inventors: 文金生
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2024-09-28
Filing date: 2024-09-28
Publication date: 2024-12-13
Anticipated expiration: 2044-09-28
Also published as: CN119119256B

Abstract

The invention discloses a monoclonal antibody of Japanese encephalitis virus and Zika virus and application thereof, and relates to the technical fields of immunology and molecular virology. The monoclonal antibody or antigen binding fragment thereof comprises a VH CDR1, a VH CDR2 and a VH CDR3 with amino acid sequences shown as SEQ ID NO 1-3 and a VL CDR1, a VL CDR2 and a VL CDR3 with amino acid sequences shown as SEQ ID NO 4-6. After a great deal of experimental research, the monoclonal antibody is found, can specifically identify and target the extracellular region of the E protein of Japanese encephalitis virus and Zika virus, can block the combination of the E protein and a cell surface receptor, and shows the capability of efficiently neutralizing viruses. Thus, the antibodies of the invention are particularly suitable for use in the diagnosis, prevention and/or treatment of encephalitis B virus and diseases associated with Zika virus infection.

Description

Monoclonal antibody of Japanese encephalitis virus and Zika virus and application thereof

Technical Field

The invention relates to the technical fields of immunology and molecular virology, in particular to a monoclonal antibody of Japanese encephalitis virus and Zika virus and application thereof.

Background

Insect-borne flaviviruses such as Japanese encephalitis virus (Japanese encephalitis virus, JEV) and Zika virus (ZIKV) that are transmitted by mosquito bites pose a serious threat to human health. The genome of this class of flaviviruses has approximately 11kb, encoding 3 structural proteins (C, prM/M and E proteins), where the E protein mediates viral infection by binding to sensitive cell surface receptors and is thus a key viral antigen that induces the production of neutralizing antibodies. ZIKV infection in adults can cause neurogenic autoimmune diseases such as Guillain Barre syndrome, and infection of pregnant women can cause neonatal microcephaly, but no Zika vaccine is currently used in batches. In addition, for JEV and ZIKV, no specific antiviral drugs or other therapies (such as antibody therapies) have been approved for clinical use.

Currently, neutralizing antibodies remain an effective method of treating viral diseases. Among the drugs that have been marketed for the treatment and prevention of viral infections are palivizumab (Synagis) for the prevention of pediatric Respiratory Syncytial Virus (RSV) infection, ai Bali beadmab (Trogarzo) for the treatment of HIV infection, and the like. Antibodies exert therapeutic effects primarily through two aspects. First, neutralizing antibodies block viral infection by binding to viral membrane proteins, blocking the binding of viruses to cellular receptors. Second, antibodies clear free viruses or kill virus-infected cells by antibody-dependent cell-mediated cytotoxicity, antibody-mediated opsonophagocytosis, activation of the complement system by antigen-antibody complexes, and the like.

Therefore, screening of broad-spectrum neutralizing monoclonal antibodies which have higher affinity and are capable of exhibiting neutralizing activity against both Japanese encephalitis virus and Zika virus has become urgent, and such antibodies can play an important role in protecting public life and health.

Disclosure of Invention

The invention aims to provide a monoclonal antibody of Japanese encephalitis virus and Zika virus and application thereof, so as to solve the problems in the prior art. The monoclonal antibody can specifically identify and target E proteins of Japanese encephalitis virus and Zika virus, particularly extracellular regions of the E proteins, and can block the combination of the E proteins and cell surface receptors, and shows the capability of efficiently neutralizing viruses.

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, the procedures of cell culture, molecular genetics, nucleic acid chemistry, immunology, virology, and the like used in the present invention, if any, are conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains [ each pair having one "light" (L) chain and one "heavy" (H) chain ]. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha or epsilon, whereby antibodies are defined as IgM, igD, igG, igA and IgE classes. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability (termed Complementarity Determining Regions (CDRs)) interspersed with regions that are more conserved, termed Framework Regions (FR). Each of VH and VL was arranged in 7 parts from amino terminal to carboxy terminal in the following order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows the definition of Kabat Sequences ofProteins of Immunological Interest【National Institutes ofHealth,Bethesda,Md.(1987and 1991)】, or Chothia et al (1989, nature. 342:878-883). The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen as the full-length antibody, and/or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion. See, generally, fundamental Immunology, ch.7 [ Paul, W., ed., 2 nd edition, RAVEN PRESS, N.Y. (1989) ], which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen binding fragments include Fab, fab ', F (ab') 2, fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (dAb), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.

In some cases, the antigen-binding fragment of an antibody is a single chain antibody (e.g., an scFv), wherein the VL and VH domains form monovalent molecules by pairing together a linker that enables production of a single polypeptide chain [ see, e.g., bird et al, 1988, science.242:423-426, and Huston et al, 1988, proc.Natl.Acad.sci.USA.85:5879-5883 ]. Such scFv molecules may have the general structure NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al, 1993, proc. Natl. Acad. Sci. USA. 90:6444-6448.). Other linkers useful in the present invention are described by Alfthan et al, 1995,Protein Eng.8:725-731.Choi et al, 2001, eur. J. Immunol.31:94-106.

In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a diabody, in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen-binding sites [ see, e.g., holliger p. Et al, 1993, proc. Natl. Acad. Sci. Usa.90:64446448, and Poljak r. J. Et al, 1994, structures.2:1121-1123 ].

The antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibody LZY2006 provided herein) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened for antibodies in the same manner as used for intact antibodies.

In the present invention, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies but also antigen-binding fragments of antibodies.

As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtainable by the hybridoma technique first reported by Kohler et al (1975, nature. 256:495), but can also be obtained by recombinant DNA techniques [ see Journal ofvirological methods,2009.158 (1-2): 171-179 ].

As used herein, a "neutralizing antibody" refers to an antibody or antibody fragment that is capable of clearing or significantly reducing the virulence (e.g., the ability to infect a cell) of a virus of interest.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as e.coli, fungal cells such as e.g. yeast cells, insect cells such as e.g. S2 drosophila cells, animal cells such as CHO cells, or human cells such as HEK293 cells, etc.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. In certain embodiments, an antibody that specifically binds (or has specificity for) an antigen refers to an antibody that binds the antigen with a dissociation equilibrium (KD) of less than about 10 ^-5 M, such as less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less.

As used herein, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding capacity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. Typically, an antibody (e.g., monoclonal antibody LZY2006 of the invention) binds an antigen (e.g., E protein of encephalitis b virus and zika virus) with a dissociation equilibrium constant (KD) of less than about 10 ^-5 M, e.g., less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less, as determined in a BIACORE 8K device using Surface Plasmon Resonance (SPR).

In the present invention, amino acids are generally indicated by single-letter or three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the maturation of virus-infected cells and/or virus progeny and/or the release of virus progeny, and an antibody or antibody fragment having neutralizing activity may prevent the amplification of a virus, thereby inhibiting or eliminating the infection by a virus.

As used herein, the term "Japanese encephalitis virus" refers to Japanese encephalitis virus (Japanese encephalitis virus, JEV) of formal classification name by the International Commission on viral Classification (International Committee on Taxonomy of Viruses, ICTV), which has the same meaning and is used interchangeably.

As used in the present invention, the terms "Japanese encephalitis" and "Japanese encephalitis" refer to encephalitis caused by JEV infection, and both have the same meaning and are used interchangeably. The term "Zika fever" refers to a disease that is mainly fever due to ZIKV infection, and both have the same meaning and are used interchangeably.

Aiming at the problems in the prior art, the invention aims to provide a monoclonal antibody with high affinity with Japanese encephalitis virus and Zika virus and application thereof.

In order to achieve the object, the invention discovers an antibody after a great deal of experimental study, which can specifically identify and target E proteins of Japanese encephalitis virus and Zika virus, especially the extracellular region of the E protein, and can block the combination of the E protein and a cell surface receptor, and shows the capability of efficiently neutralizing the virus. Thus, the antibodies of the invention are particularly suitable for use in diagnosing, preventing and/or treating encephalitis B virus and diseases associated with Zika virus infection (e.g., encephalitis B and Zika fever).

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a monoclonal antibody or antigen binding fragment thereof of Japanese encephalitis virus and Zika virus, which comprises a VH CDR1, a VH CDR2 and a VH CDR3 with amino acid sequences shown as SEQ ID NO 1-3, and a VL CDR1, a VL CDR2 and a VL CDR3 with amino acid sequences shown as SEQ ID NO 4-6.

Further, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

In certain preferred embodiments, the monoclonal antibody further has a signal peptide at the N-terminus of the heavy chain variable region. In certain preferred embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 11 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 17.

In certain preferred embodiments, the monoclonal antibody further has a signal peptide at the N-terminus of the light chain variable region. In certain preferred embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 12 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 18.

In certain preferred embodiments, the monoclonal antibodies further comprise a heavy chain constant region. In certain preferred embodiments, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO. 9. The gene sequence of the heavy chain constant region is shown as SEQ ID NO. 15.

In certain preferred embodiments, the monoclonal antibody further comprises a light chain constant region. In certain preferred embodiments, the amino acid sequence of the light chain constant region is set forth in SEQ ID NO. 10. The gene sequence of the light chain constant region is shown as SEQ ID NO. 16.

In certain preferred embodiments, the light chain of the monoclonal antibody is kappa-type.

In certain preferred embodiments, the monoclonal antibody or antigen binding fragment thereof is capable of specifically binding to envelope proteins (E proteins) of encephalitis b virus and zika virus. In certain preferred embodiments, the monoclonal antibody or antigen binding fragment thereof is capable of targeting the extracellular region of the E protein of encephalitis b virus and zika virus. In certain preferred embodiments, the monoclonal antibody or antigen binding fragment thereof is capable of inhibiting extracellular domain-mediated receptor binding and/or membrane fusion processes of the E protein, inhibiting viral infection of cells.

In certain preferred embodiments, the monoclonal antibody or antigen binding fragment thereof has neutralizing capacity (e.g., is capable of neutralizing encephalitis b virus and zika virus). In certain preferred embodiments, the monoclonal antibody or antigen binding fragment thereof is capable of inhibiting infection by encephalitis B virus and Zika virus or entry into a host cell. Thus, the monoclonal antibody or antigen binding fragment thereof is capable of neutralizing Japanese encephalitis virus and Zika virus, and thereby preventing and treating diseases associated with Japanese encephalitis virus and Zika virus infection.

The invention also provides a coding gene of the monoclonal antibody or the antigen binding fragment thereof.

Preferably, the coding gene comprises a DNA molecule with the nucleotide sequences shown in SEQ ID NO. 13 and SEQ ID NO. 14. The DNA molecule shown in SEQ ID NO. 13 is capable of encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, and the DNA molecule shown in SEQ ID NO. 14 is capable of encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

In certain preferred embodiments, the coding gene further comprises a DNA molecule encoding a signal peptide sequence located 5' to the DNA molecule capable of encoding the heavy chain variable region of the monoclonal antibody or antigen binding fragment thereof of the invention. In certain preferred embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO. 11. In certain preferred embodiments, the DNA molecule encoding the signal peptide has the nucleotide sequence set forth in SEQ ID NO. 17.

In certain preferred embodiments, the DNA molecule further comprises a nucleotide sequence encoding a signal peptide located 5' to the DNA molecule capable of encoding the light chain variable region of the monoclonal antibody or antigen binding fragment thereof of the invention. In certain preferred embodiments, the signal peptide has the amino acid sequence shown in SEQ ID NO. 12. In certain preferred embodiments, the DNA molecule encoding the signal peptide sequence has the nucleotide sequence set forth in SEQ ID NO. 18.

In certain preferred embodiments, the DNA molecule comprises a first polynucleotide comprising a nucleotide sequence encoding a signal peptide and a nucleotide sequence capable of encoding a heavy chain variable region of a monoclonal antibody or antigen binding fragment thereof of the invention, and a second polynucleotide comprising a nucleotide sequence encoding a signal peptide and a nucleotide sequence capable of encoding a light chain variable region of a monoclonal antibody or antigen binding fragment thereof of the invention.

In certain preferred embodiments, the coding gene comprises a first polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO. 17 and the nucleotide sequence set forth in SEQ ID NO. 13, and a second polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO. 18 and the nucleotide sequence set forth in SEQ ID NO. 14.

In certain preferred embodiments, the coding gene further comprises a nucleotide sequence capable of encoding a heavy chain constant region of a monoclonal antibody or antigen binding fragment thereof of the invention. In certain preferred embodiments, the heavy chain constant region has the amino acid sequence set forth in SEQ ID NO. 9. In certain preferred embodiments, the nucleotide sequence capable of encoding the heavy chain constant region of a monoclonal antibody or antigen binding fragment thereof of the invention has the nucleotide sequence set forth in SEQ ID NO. 15.

In certain preferred embodiments, the coding gene further comprises a nucleotide sequence capable of encoding a light chain constant region of a monoclonal antibody or antigen binding fragment thereof of the invention. In certain preferred embodiments, the light chain constant region has the amino acid sequence set forth in SEQ ID NO. 10. In certain preferred embodiments, the nucleotide sequence capable of encoding the light chain constant region of the monoclonal antibodies or antigen binding fragments thereof of the invention has the nucleotide sequence set forth in SEQ ID NO. 16.

In certain preferred embodiments, the coding gene comprises a first polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence, a nucleotide sequence capable of encoding a heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof of the invention, and a nucleotide sequence capable of encoding a heavy chain constant region of a monoclonal antibody or antigen-binding fragment thereof of the invention, and a second polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence, a nucleotide sequence capable of encoding a light chain variable region of a monoclonal antibody or antigen-binding fragment thereof of the invention, and a nucleotide sequence capable of encoding a light chain constant region of a monoclonal antibody or antigen-binding fragment thereof of the invention.

In certain preferred embodiments, the coding gene comprises a first polynucleotide comprising the nucleotide sequences set forth in SEQ ID NO. 17, SEQ ID NO. 13 and SEQ ID NO. 15, and a second polynucleotide comprising the nucleotide sequences set forth in SEQ ID NO. 18, SEQ ID NO. 14 and SEQ ID NO. 16.

The invention also provides a recombinant vector comprising the coding gene. The vector of the present invention may be a cloning vector or an expression vector. In certain preferred embodiments, the vectors of the present invention are, for example, plasmids, cosmids, phages and the like.

The invention also provides a recombinant host cell comprising the coding gene or the recombinant vector. Such host cells include, but are not limited to, prokaryotic cells, such as E.coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). The cells of the invention may also be cell lines, such as 293T cells.

The invention also provides application of the coding gene, the recombinant vector or the recombinant host cell in preparing monoclonal antibodies or antigen binding fragments thereof of Japanese encephalitis virus and Zika virus.

The invention also provides a method of preparing a monoclonal antibody or antigen-binding fragment thereof of the invention, comprising the steps of culturing a recombinant host cell of the invention under suitable conditions, and recovering the monoclonal antibody or antigen-binding fragment thereof of the invention from the cell culture.

The present invention also provides the use of the monoclonal antibody or antigen binding fragment thereof described above in any one of the following (1) - (3):

(1) Preparing a product for detecting Japanese encephalitis virus, japanese encephalitis virus E protein extracellular domain or Japanese encephalitis virus E protein extracellular domain;

(2) Preparing a product for neutralizing the virulence of Japanese encephalitis virus or Zika virus;

(3) Preparing medicine for preventing and/or treating encephalitis B virus and/or diseases related to Zika virus infection.

The invention also provides a product for detecting Japanese encephalitis virus, zika virus, japanese encephalitis virus E protein, zika virus E protein, japanese encephalitis virus E protein extracellular region or Zika virus E protein extracellular region, which comprises the monoclonal antibody or antigen binding fragment thereof.

In certain preferred embodiments, the product further comprises a second antibody that specifically recognizes a monoclonal antibody or antigen-binding fragment thereof or an anti-idiotype antibody of the invention. Preferably, the second antibody further comprises a detectable label. Such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials, enzymes (e.g., horseradish peroxidase), and the like.

The invention also provides a pharmaceutical composition comprising pharmaceutically acceptable auxiliary materials and the monoclonal antibody or the antigen binding fragment thereof.

In certain preferred embodiments, the adjuvant is a carrier and/or excipient.

Preferably, the pharmaceutical composition further comprises other pharmaceutically active agents, such as ribavirin and the like.

The invention discloses the following technical effects:

The monoclonal antibody LZY2006 provided by the invention can be combined with the extracellular region of E protein of Japanese encephalitis virus and Zika virus with high affinity, and has strong neutralization activity on Japanese encephalitis virus and Zika virus, so that the monoclonal antibody is used as a broad-spectrum neutralization antibody. The monoclonal antibody LZY2006 of the invention has ideal clinical application value for preventing and treating Japanese encephalitis virus and Zika virus infection, and is expected to play an important role in protecting public life and health.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a SDS-PAGE electrophoresis of a monoclonal antibody LZY2006 expressed in example 4 of the present invention, wherein "no DTT" on the gel indicates no DTT added (non-reducing SDS-PAGE) and "DTT" indicates DTT added (reducing SDS-PAGE);

FIG. 2 is a graph showing the kinetics of binding of LZY2006 monoclonal antibody at different concentrations to the E protein extracellular domain (A) of Japanese encephalitis virus and the E protein extracellular domain (B) of Zika virus in example 5 of the present invention, wherein in A and B, the abscissa indicates time (seconds) and the ordinate indicates response value (RU);

FIG. 3 is a graph showing the results of in vitro neutralization activity detection of LZY2006 monoclonal antibodies of different concentrations in example 6 of the present invention on Japanese encephalitis virus and Zika virus, wherein the ordinate Neutralization (%) represents the neutralization percentage, and the abscissa represents the concentration;

FIG. 4 shows the results of survival experiments of mice protected against lethal Japanese encephalitis virus and Zika virus at different doses of LZY2006 mAb of example 7 of the present invention, wherein A is the percentage of body weight to original body weight of mice given with 1 μg of LZY2006 mAb under JEV (10 PFU) infection, B is the percentage of body weight of mice given with 1 μg of LZY2006 mAb under JEV (10 PFU) infection, C is the percentage of body weight to original body weight of mice given with 25 μg of LZY2006 mAb under JEV (10 PFU) infection, D is the percentage of body weight to original body weight of mice given with 25 μg of LZY2006 mAb under JEV (10 PFU) infection, E is the percentage of body weight to original body weight of mice given with ZIKV (1000 PFU) infection, F is the percentage of body weight to ZIZ (1000 PFU) infection, C is the percentage of body weight to 1 μg of mice given with LZY2006 mAb under ZIV (1000 PFU) infection, D is the percentage of body weight to 25 μg 2006% of body weight to the mice given with 25 KV (25 KV) infection, and the percentage of body weight to the mice after 25 KV (25 KV) infection, and the percentage of body weight to which is 25 KV is given in the days after the mice are given.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

Unless otherwise indicated, the molecular biology experimental methods and immunoassays used in the present invention are essentially described in J.Sambrook et al, molecular cloning, in laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-programming molecular biology experimental guidelines, 3 rd edition, john Wiley & Sons, inc.,1995, and the use of restriction enzymes is in accordance with conditions recommended by the manufacturer of the product. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The information of the partial sequences according to the present invention is shown in table 1 below.

TABLE 1 amino acid and nucleotide sequences

In order to obtain the neutralizing antibody with protective effect, the invention firstly takes the extracellular region (E410) of E protein of the Zika virus expressed by escherichia coli as an antigen, screens out memory B cells capable of specifically binding ZIKV-E410 from Peripheral Blood Mononuclear Cells (PBMCs) of adult healthy volunteers (22 years old) inoculated with encephalitis B virus attenuated vaccine by a flow sorting technique, and then performs BCR 10X Genomics sequencing on single B cells obtained by screening to obtain the gene sequence encoding the antibody variable region in the single B cells. Further, a sequence encoding the antibody variable region and a constant region gene are ligated into an expression vector, and expressed and purified in mammalian cells, thereby obtaining mab LZY2006. A series of functional tests are carried out on the monoclonal antibody LZY2006, and the result shows that the monoclonal antibody LZY2006 can specifically bind to JEV-E406 and ZIKV-E410, inhibit infection of Japanese encephalitis virus and Zika virus to Vero cells, protect mice against lethal Japanese encephalitis virus and Zika virus attack, and has broad-spectrum neutralization activity against Japanese encephalitis virus and Zika virus infection. The specific details are as follows:

Example 1 expression and purification of E protein extracellular regions of Japanese encephalitis Virus and Zika Virus

1. The coding gene sequences of JEV-E406 (residues 1-406,GenBank accession no:MK558811) and ZIKV-E410 (residues 1-410,GenBank accession no:KX117076.1) with 6 His labels at the C-terminal after the optimization of the escherichia coli codons are cloned into a prokaryotic expression vector pET21a respectively to obtain recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410.

2. Recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410 were transformed into E.coli BL21 strain respectively and the protein was expressed by IPTG induction.

3. JEV-E406 and ZIKV-E410 proteins are separated and purified by utilizing a Ni-NTA affinity chromatographic column. SDS-PAGE and WB experiments were used to detect the purity of both proteins.

Example 2 isolation of memory B cells specifically recognizing ZIKV-E410 protein

1. 100ML of peripheral venous blood was collected under informed consent from healthy volunteers vaccinated with JEV attenuated live vaccine, and Peripheral Blood Mononuclear Cells (PBMC) were isolated using human lymphocyte isolates.

2. The isolated PBMCs were combined with biotinylated antibody mixtures (containing biotinylated anti-human CD2 mab, anti-human CD3 mab, anti-human CD14 mab, anti-human CD43 mab, anti-human CD56 mab, anti-human CD235a mab) from maytansinol (Miltenyi) in flow tubes followed by incubation at 4 ℃ for 30min. Adding pre-chilled buffer (0.5% BSA/PBS containing 20mM EDTA), adding Anti-biotin microbeads, incubating at 4deg.C for 30min, adding pre-chilled buffer (0.5% BSA/PBS containing 20mM EDTA), centrifuging at 2000rpm for 10min, discarding supernatant, adding pre-chilled buffer (0.5% BSA/PBS containing 20mM EDTA) to suspend cell pellet, transferring into flow tube, inserting magnet for 3min, collecting non-adsorbed cells, washing adsorbed magnetic beads with pre-chilled buffer (0.5% BSA/PBS containing 20mM EDTA), inserting magnet for 3min, collecting non-adsorbed cells, transferring all non-adsorbed cell liquid into 15mL centrifuge tube, centrifuging at 2000rpm for 10min, discarding supernatant, and suspending cell (memory B cell) with pre-chilled buffer (0.5% BSA/PBS containing 20 mM).

3. Adding 5mL of magnetic beads (1 μm diameter) into a flow tube, inserting a magnet for 3min, discarding supernatant, adding 4mL of PBS (phosphate buffer solution) for 3min, inserting a magnet for 3min, discarding supernatant, adding 4mL of 5 mu M imidazole solution, inserting a magnet for 3min, discarding supernatant, adding protein solution (2 mg ZIKV-E410) and a proper amount of binding buffer solution to 4mL, screwing a cover onto a shaker for 40min, inserting a magnet for 3min, discarding supernatant, adding 4mLPBS of the PBS (phosphate buffer solution), inserting a magnet for 3min, discarding supernatant, adding 4mL of 5% BSA/PBS (phosphate buffer solution) for 10min, inserting a magnet for 3min, discarding supernatant, adding 4mLPBS of the PBS (buffer solution), inserting a magnet for 3min, discarding supernatant, suspending the magnetic beads with 1mL of memory B cell suspension (separated from 100mL peripheral venous blood), shaking up for 20min, adding unbound buffer solution for 4mL, inserting a magnet for 3min, discarding unbound buffer solution for 3min, and pre-cooling the cell under pre-cooling (target cell washing).

4. The cells were sent to the company for 10×single cell expression profiling and BCR library sequencing and analysis.

Example 3 isolation and identification of LZY2006 mab and construction of recombinant expression vectors

The gene of the V region of BCR of more than 6000 memory B cells is detected altogether, and the sequence analysis is carried out, so that the invention selects a variable region (V region) sequence of a light chain and a heavy chain named LZY2006 monoclonal antibody for subsequent research.

The amino acid sequence of the heavy chain variable region of LZY2006 monoclonal antibody is shown as SEQ ID NO. 7 (the coding gene is shown as SEQ ID NO. 13), the amino acid sequence of the VH CDR1 of the heavy chain variable region is shown as SEQ ID NO. 1, the amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 3. The amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8 (the coding gene is shown as SEQ ID NO. 14), the amino acid sequence of the VL CDR1 of the light chain variable region is shown as SEQ ID NO. 4, the amino acid sequence of the VL CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO. 6. The V region genes for the LZY2006 monoclonal antibodies heavy and light chains are shown in tables 2-3.

TABLE 2LZY2006 Single heavy chain V region Gene

Monoclonal antibody	V-H alleles	J-H alleles
			LZY2006	IGHV4-59	IGHJ6

TABLE 3LZY2006 monoclonal antibody light chain V region Gene

Monoclonal antibody	V-L alleles	J-L alleles
			LZY2006	IGLV3-25	IGLJ2

The nucleotide sequences encoding the heavy and light chain variable regions of LZY2006 obtained by the analysis were placed in the corresponding expression vectors pCAGGS (present laboratory construction) containing the nucleotide sequences encoding the constant regions of the heavy/kappa chains, respectively, to obtain recombinant expression vectors pCAGGS-LZY2006H and pCAGGS-LZY2006L encoding the single heavy and light chains of LZY2006, respectively.

Heavy chain coding sequence (5 '-3')CMVpromoter-EcoR I cleavage site-signal peptide sequence gene-VH gene-CH gene-Xho I cleavage site;

The light chain (kappa) coding sequence (5 '-3') CMV promoter-EcoR I cleavage site-signal peptide sequence gene-VL gene-CL (kappa) gene-Xho I cleavage site;

Wherein the amino acid sequence of the heavy chain coding sequence signal peptide sequence is shown as SED ID NO. 11 (the nucleotide sequence of the coding gene is shown as SEQ ID NO. 17), the amino acid sequence of the light chain coding sequence signal peptide sequence is shown as SED ID NO. 12 (the nucleotide sequence of the coding gene is shown as SEQ ID NO. 18);

The nucleotide sequence of the VH gene is shown as SEQ ID NO. 13, and the nucleotide sequence of the VL gene is shown as SEQ ID NO. 14;

the amino acid sequence of CH is shown as SED ID NO. 9 (the nucleotide sequence of the coding gene is shown as SEQ ID NO. 15), the amino acid sequence of CL is shown as SED ID NO. 10 (the nucleotide sequence of the coding gene is shown as SEQ ID NO. 16).

Example 4 expression of LZY2006 mab

The heavy and light chain plasmids (recombinant expression vectors pCAGGS-LZY2006H and pCAGGS-LZY2006L encoding the LZY2006 monoclonal antibody heavy and light chain, respectively, obtained in example 3) were co-transfected into 293T cells. The weight ratio of the heavy chain plasmid pCAGGS-LZY2006H to the light chain plasmid pCAGGS-LZY2006L was 1.07:0.93, and 2. Mu.g of plasmid (heavy chain plasmid+light chain plasmid) and 4. Mu.g of PEI were transfected into 293T cells per cell plate (10 cm in diameter). Cell supernatants were collected 48h and 96h post-transfection and filtered through a 0.22 μm needle filter, then purified using a Pierce protein A/G agarose column (Thermo Fisher) and the mAb concentrated using a 50kDa cut-off molecular weight ultrafiltration tube. Subsequently, electrophoresis was run by SDS-PAGE (in both reducing and non-reducing states). The results are shown in fig. 1, and purified LZY2006 mab was obtained.

Example 5 evaluation of the ability of LZY2006 mab to bind to JEV-E406 and ZIKV-E410

In this example, a LIFEDISCTM METASPR chip (Metro Hangzhou Biotechnology Co., ltd.) was used for surface plasmon resonance analysis. The method comprises the following specific steps:

first, JEV-E406 or ZIKV-E410 (20. Mu.g/mL) was immobilized on LIFEDISCTM METASPR chip (Quantum [ Hangzhou ] Biotechnology Co., ltd.), and LZY2006 diluted with a continuous multiple ratio of PBST solution at pH 7.4 was sequentially loaded one by one through each channel (40, 80, 160, 320. Mu.g/mL [ i.e., 0.267. Mu.M, 0.534. Mu.M, 1.068. Mu.M, 2.136. Mu.M ]. Kinetic curves of LZY3412 binding to JEV-E406 or ZIKV-E410 proteins were recorded (shown in FIG. 2) and fitted in a "1:1binding" mode using WeSPR One software (amount of Qmotion Biotech Co., ltd.) to calculate kinetic constants (shown in Table 4). The results in FIG. 2 and Table 4 show that LZY2006 mab is capable of binding with high affinity to the E protein extracellular domains of JEV and ZIKV (JEV-E406 and ZIKV-E410). Wherein the amino acid sequence of JEV-406 and the amino acid sequence of ZIKV-E410 are shown in Table 1.

Table 4 affinity of LZY2006 mab to JEV-E406 and ZIKV-E410

Example 6LZY2006 Single antibody Ex-neutralization JEV and ZIKV experiments

1. Vero cells were plated on 24 well plates (1×10 ⁵/well) and cultured for 24h.

2. LZY2006 mab to be tested was diluted 3-fold in 96-well plates (starting from 3.67. Mu.g/mL), virus to be tested (JEV or ZIKV,50 virus Plaque Forming Units (PFU)/well) was added to each well and incubated at 37℃for 1h.

3. The Vero cell supernatant was discarded and the virus/mab mixture was added to the cells and infected at 37 ℃ for 1.5h.

4. The virus solution is discarded, 1% methyl cellulose culture solution is added to each hole to cover the cells, and the cells are cultured for 3 to 6 days.

5. 4% Paraformaldehyde is added into each hole to be fixed for 1h at room temperature, and the plate is washed.

6. And adding 0.5% crystal violet dye solution into each hole, dyeing for 10min at room temperature, and washing the plate.

7. The number of plaques per well of virus was counted and the neutralization titer (half inhibitory concentration, IC ₅₀ value) of diluted mab for neutralization of each virus was calculated.

IC ₅₀ was analyzed using GRAPHPAD PRISM 6 software. The analysis results are shown in FIG. 3 and Table 5. From this, LZY2006 mab was able to inhibit JEV and ZIKV with extremely high neutralizing activity.

Table 5 neutralizing titres of LZY2006 mab to JEV and ZIKV (semi-inhibitory concentration, IC ₅₀)

Example 7 experiments with LZY2006 monoclonal antibody protecting mice against lethal JEV or ZIKV challenge

1. Injecting JEV (10 PFU) or ZIKV (1000 PFU) into the back of a 1-day-old mouse subcutaneously;

after 2.2h, mice were injected subcutaneously with 1 μg or 25 μg of LZY2006 mab into their backs for survival experiments (mice weight and death were recorded daily).

3. Data were analyzed, counted and plotted using Prism 6, and the results are shown in fig. 4.

The results showed that the percent survival of mice given 1 μg or 25 μg of LZY2006 mab after JEV infection was significantly higher than the percent survival of mice not given LZY2006 mab (16.7%versus 0,P<0.0001;42.9%versus 0,P =0.0001, respectively), and that the percent survival of mice given 25 μg of LZY2006 mab after ZIKV infection was significantly higher than the percent survival of mice not given LZY2006 mab (50% of versus 0, p=0.0011).

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof against Japanese encephalitis virus and Zika virus, characterized in that it comprises VH CDR1, VH CDR2 and VH CDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3, and VL CDR1, VL CDR2 and VL CDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

3. A gene encoding the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2.

4. The coding gene according to claim 3 is characterized in that it comprises a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 13 and/or SEQ ID NO: 14.

5. A recombinant vector, characterized in that it comprises the coding gene according to claim 3 or 4.

6. A recombinant host cell, characterized in that it comprises the encoding gene according to claim 3 or 4 or the recombinant vector according to claim 5.

7. Use of the encoding gene according to claim 3 or 4, the recombinant vector according to claim 5 or the recombinant host cell according to claim 6 in preparing monoclonal antibodies or antigen-binding fragments thereof against Japanese encephalitis virus and Zika virus.

8. Use of the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2 in any one of the following (1) to (3):

(1) Preparation of products for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, Japanese encephalitis virus E protein extracellular region or Zika virus E protein extracellular region;

(2) Preparation of products for neutralizing the virulence of Japanese encephalitis virus or Zika virus;

(3) Preparing a drug; the drug is used to prevent and/or treat diseases related to Japanese encephalitis virus and/or Zika virus infection.

9. A product for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, Japanese encephalitis virus E protein extracellular region or Zika virus E protein extracellular region, characterized in that it comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

10. A pharmaceutical composition, characterized in that it comprises a pharmaceutically acceptable excipient and the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.