Broad spectrum binding monoclonal antibody targeting alpha herpes virus and application thereof

Abstract

The invention relates to the fields of immunology and molecular virology, in particular to the field of alpha herpes virus diagnosis. Specifically, the invention relates to an anti-alpha herpes virus monoclonal antibody and a kit containing the antibody. In addition, the invention also relates to application of the antibody.

Description

Technical Field

[0001] The present invention relates to the field of immunology and molecular virology, in particular to the field of diagnosis of alpha herpes virus. Specifically, the present invention relates to monoclonal antibodies against alpha herpes virus, and kits comprising the antibodies. In addition, the present invention also relates to the use of the antibodies. Background Art

[0002] The herpesvirus family can be divided into three subfamilies: α, β and γ: ① α subfamily, represented by herpes simplex virus, with a small DNA molecular weight and fast replication; ② β subfamily, represented by human cytomegalovirus, with a large DNA molecular weight and slow replication; ③ γ subfamily, represented by Epstein-Barr (EB) virus, characterized by infecting lymphocytes. Among them, the α herpesvirus subfamily can be divided into the herpes simplex virus genera, varicella virus genera, laryngovirus genera and Malik virus genera. The main representative species of the herpes simplex virus genus are human herpes simplex virus type 1, human herpes simplex virus type 2, monkey B virus, and bovine herpes virus type 2; the main representative species of the varicella virus genus are human herpes virus type 3, pseudorabies virus (PRV), bovine herpes virus type 1, and bovine herpes virus type 5; the main representative species of the laryngovirus genus is avian herpes virus type 1; the main representative species of the Malik virus genus are avian herpes virus type 2 and duck herpes virus type 1.

[0003] Herpes simplex virus and varicella virus mainly infect mammals, while herpes simplex virus and malik virus mainly infect birds. Some viruses in the herpes simplex virus and varicella virus genera are extremely harmful to humans.

[0004] Herpes simplex virus and simian B virus are typical representatives of the herpes simplex virus genus. Herpes simplex virus is named because of the vesicular dermatitis, i.e. herpes simplex, that occurs during the acute phase of infection. It can cause a variety of human diseases, such as gingivostomatitis, keratoconjunctivitis, encephalitis, as well as reproductive system infections and neonatal infections. According to WHO statistics, in 2012, 3.709 billion people aged 0 to 49 worldwide had oral herpes due to infection with HSV-1, accounting for 67% of the global population, and the prevalence rate increased with age. Another statistical analysis in 2016 found that 13.2% of the world's population (about 490 million people) had genital herpes due to infection with HSV-2. In sub-Saharan Africa and Latin America, the serum positivity rate of many populations exceeds 90%. In many areas, the HSV infection rate of people over 50 years old is close to 100%. The recurrence rate of genital herpes caused by HSV-2 is significantly higher than that of HSV-1, about 6 times that of HSV-1. HSV increases the susceptibility and infectiousness of HIV, and can increase the risk of new HIV infection by about 3 times. People with both HIV and HSV infection are more likely to transmit HIV to others. In the late stage of HIV infection, HSV can lead to more serious complications, such as hemorrhagic necrotizing encephalitis, esophagitis, hepatitis, pneumonia, retinal necrosis or disseminated infection.

[0005] Monkey B virus is an endemic alpha herpes virus in macaques, usually transmitted horizontally through direct contact and exchange of body secretions. The pathogenesis of monkey B virus infection is similar to that of HSV (herpes simplex virus). The virus first proliferates in epithelial tissues and then invades nerve cells. Generally, monkey B virus neutralizing antibodies appear in the serum 3 weeks after infection. Pathological changes include blisters on the back of the tongue, and mucosal blisters on the edges of the lips or other cavities gradually rupture and blister marks appear. Generally, they heal after 2 weeks. At the same time, monkeys may also develop severe keratitis. This virus is not obvious in the natural host macaques, but there have been about 60 sporadic cases of pathogenic zoonotic monkey B virus infection, with a mortality rate of 70%-80%, which is higher than the 50% mortality rate of Ebola virus.

[0006] Human herpesvirus type 3 and pseudorabies virus (PRV) are typical representatives of the genus Varicella virus. Human herpesvirus type 3 is also called varicella-zoster virus (VZV). VZV is a widespread and highly contagious human alpha-herpes virus. The first infection with VZV can cause chickenpox, and the general population is susceptible (the infection rate is about 61% to 100%). The virus can be latent in the dorsal root ganglia and persist for life. Varicella has caused a huge disease burden worldwide, with at least 140 million new cases of varicella, 4.2 million cases of severe complications of varicella, and 4,200 deaths each year. In addition, nearly one-third of VZV-infected people will develop herpes zoster in old age due to the reactivation of VZV lurking in the nerves, which is usually accompanied by severe neuralgia. Even after recovery, postherpetic neuralgia will occur, seriously affecting the patient's quality of life. Varicella and herpes zoster can also cause serious life-threatening complications in newborns and immunocompromised people.

[0007] PRV causes acute infectious diseases in a variety of livestock and wild animals, characterized by fever, itching and encephalomyelitis. Pigs are the natural hosts of PRV. PRV infection in pigs can cause severe viral encephalitis, respiratory distress, growth retardation in growing and fattening pigs, reproductive disorders in sows and death of newborn piglets, causing serious economic losses to the global pig industry. In addition, the harm of PRV to human health cannot be ignored. In recent years, reports of viral encephalitis caused by PRV crossing the species barrier to infect humans indicate that the virus has created a new disease burden.

[0008] All of the above viruses can cause serious harm to human health, and mixed infection with multiple viruses may lead to more serious harm. However, there is currently a lack of efficient diagnostic reagents for mixed infection with multiple herpes viruses. Therefore, it is of great significance to develop a broad-spectrum binding antibody that can simultaneously target the above viruses as a diagnostic reagent for herpes virus-related diseases. Summary of the invention

[0009] The inventors of the present application have obtained, after a large number of experiments and repeated explorations, monoclonal antibodies with broad-spectrum binding activity to a variety of alpha herpes viruses for the first time. Therefore, the antibodies of the present invention have the potential to be used for diagnosing herpes virus infection or diseases caused by herpes virus infection, and have great clinical value.

[0010] Antibodies of the present invention

[0011] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to herpes virus gB protein, wherein the antibody or the antigen-binding fragment thereof comprises:

[0012] (a) a heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VH CDR1 of SEQ ID NO: 3, VH CDR2 of SEQ ID NO: 4, and VH CDR3 of SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 of SEQ ID NO: 6, VL CDR2 of SEQ ID NO: 7, and VL CDR3 of SEQ ID NO: 8; or

[0013] (b) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 19, VH CDR2 of SEQ ID NO: 20, and VH CDR3 of SEQ ID NO: 21; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 22, VL CDR2 of SEQ ID NO: 23, and VL CDR3 of SEQ ID NO: 24.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0015] (a) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1 or a variant thereof; and / or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or,

[0016] (b) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 17 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 18 or a variant thereof.

[0017] Wherein, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO: 1, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto, and a VL comprising a sequence as shown in SEQ ID NO: 2, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO: 1, and a VL comprising a sequence as shown in SEQ ID NO: 2.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO: 17, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto, and a VL comprising a sequence as shown in SEQ ID NO: 18, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO: 17, and a VL comprising a sequence as shown in SEQ ID NO: 18.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (eg, murine or human) immunoglobulin.

[0021] In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0022] In certain embodiments, the light chain constant region is a kappa or lambda light chain constant region.

[0023] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a murine or human immunoglobulin (eg, IgG, such as IgG1, IgG2, IgG3, or IgG4).

[0024] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a murine or human immunoglobulin (eg, kappa or lambda, eg, kappa).

[0025] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:9 and / or a light chain constant region (CL) as shown in SEQ ID NO:10.

[0026] In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise: a heavy chain comprising VH as shown in SEQ ID NO:1 and CH as shown in SEQ ID NO:9, and / or a light chain comprising VL as shown in SEQ ID NO:2 and CL as shown in SEQ ID NO:10.

[0027] In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise: a heavy chain comprising VH as shown in SEQ ID NO:17 and CH as shown in SEQ ID NO:9, and / or a light chain comprising VL as shown in SEQ ID NO:18 and CL as shown in SEQ ID NO:10.

[0028] In certain embodiments, the antigen binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb). In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, a bispecific antibody or a multispecific antibody.

[0029] In certain embodiments, the antibody or its antigen-binding fragment can specifically bind to alpha herpes virus gB protein. In certain embodiments, the alpha herpes virus is selected from the genus Herpes simplex virus and / or the genus Varicella herpes virus. In certain embodiments, the alpha herpes virus is selected from HSV-1, HSV-2, monkey B virus, PRV, VZV or any combination thereof. In certain embodiments, the antibody or its antigen-binding fragment can specifically bind to a target protein selected from the following: gB protein of HSV-1 (e.g., comprising an extracellular domain as shown in SEQ ID NO: 13), gB protein of HSV-2 (e.g., comprising an extracellular domain as shown in SEQ ID NO: 25), gB protein of monkey B virus (e.g., comprising an extracellular domain as shown in SEQ ID NO: 14), gB protein of PRV (e.g., comprising an extracellular domain as shown in SEQ ID NO: 15), gB protein of VZV (e.g., comprising an extracellular domain as shown in SEQ ID NO: 16).

[0030] Herein, the antibodies or antigen-binding fragments thereof of the present invention may include variants that differ from the antibodies or antigen-binding fragments thereof from which they are derived only in conservative substitutions of one or more (e.g., up to 20, up to 15, up to 10, or up to 5) amino acid residues, or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the antibodies or antigen-binding fragments thereof from which they are derived, and substantially retain the above-mentioned biological functions of the antibodies or antigen-binding fragments thereof from which they are derived (e.g., broad-spectrum binding activity to herpes viruses, particularly alpha herpes viruses).

[0031] Antibody preparation

[0032] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present invention.

[0033] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab' fragments can be directly obtained from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). In addition, Fv, Fab or F(ab')2 fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0034] Therefore, in another aspect, the invention provides a kind of isolated nucleic acid molecule, it comprises the nucleotide sequence of encoding antibody of the present invention or its Fab, or its heavy chain variable region and / or light chain variable region.In certain embodiments, the isolated nucleic acid molecule encodes antibody of the present invention or its Fab, or its heavy chain variable region and / or light chain variable region.

[0035] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or its antigen-binding fragment of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or its antigen-binding fragment, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule containing the first nucleotide sequence and a second nucleic acid molecule containing the second nucleotide sequence.

[0036] In another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, and the like.

[0037] In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or an antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region of the antibody or an antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0038] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention, and / or a second nucleotide sequence encoding the light chain variable region of an antibody or antigen-binding fragment thereof of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0039] In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain of an antibody or antigen-binding fragment thereof of the present invention, and / or a second nucleotide sequence encoding the light chain of an antibody or antigen-binding fragment thereof of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0040] In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell of the present invention is a mammalian cell, such as a CHO cell.

[0041] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, which comprises culturing the host cell as described above under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0042] Conjugate

[0043] The antibody or its antigen-binding fragment of the present invention can be derivatized, for example, connected to another molecule (e.g., another polypeptide or protein). Generally, the derivatization (e.g., labeling) of the antibody or its antigen-binding fragment will not adversely affect its binding to the antigen (e.g., herpes virus gB protein). Therefore, the antibody or its antigen-binding fragment of the present invention is also intended to include such derivatized forms. For example, the antibody or its antigen-binding fragment of the present invention can be functionally connected (by chemical coupling, gene fusion, non-covalent connection or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide (e.g., avidin or polyhistidine tag) that can mediate the binding of the antibody or antigen-binding fragment to another molecule. In addition, the antibody or its antigen-binding fragment of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.

[0044] Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof of the invention are detectably labeled.

[0045] In this context, the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0046] In certain embodiments, the detectable label can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable label can be selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase or β-galactosidase), chemiluminescent agents (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent proteins, such as FITC, TRITC or PE), radionuclides or biotin.

[0047] In certain embodiments, the detectable labels described above can be linked to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

[0048] Kit and test purpose

[0049] The antibody or antigen-binding fragment thereof of the present invention can specifically bind to the gB protein of herpes virus, and thus can be used to detect herpes virus or its gB protein, and optionally diagnose whether the subject is infected with herpes virus according to the above detection results. In particular, the antibody or antigen-binding fragment thereof of the present invention has a broad-spectrum binding activity to multiple alpha herpes viruses, thereby facilitating the detection and diagnosis of mixed infection of multiple alpha herpes viruses.

[0050] Therefore, in another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention or the conjugate of the present invention.

[0051] In some embodiments, the kit comprises a conjugate of the invention.

[0052] In other embodiments, the kit comprises an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the antibody or antigen-binding fragment thereof does not comprise a detectable label. In certain embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0053] In certain embodiments, the second antibody is specific for an antibody of the species (eg, mouse or human) from which the constant region comprised by the antibody or antigen-binding fragment thereof of the invention is derived.

[0054] In certain embodiments, the second antibody is an anti-immunoglobulin (e.g., mouse or human immunoglobulin) antibody, such as an anti-IgG antibody. In certain embodiments, the second antibody is an anti-human IgG antibody. In certain embodiments, the second antibody is an anti-mouse IgG antibody.

[0055] In certain embodiments, the kit of the present invention may further comprise a reagent for detecting the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may further comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS or luminol compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., an acridinium ester compound), the kit may further comprise a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0056] In another aspect, the present invention provides a method for detecting the presence or level of herpes virus (or its gB protein, or cells infected by herpes virus) in a sample, comprising using the antibody or antigen-binding fragment thereof, conjugate or kit of the present invention.

[0057] In certain embodiments, the method is an immunological assay, such as an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0058] In some embodiments, the method comprises using a conjugate of the invention.

[0059] In other embodiments, the method comprises using an antibody or antigen-binding fragment thereof of the present invention. In certain embodiments, the antibody or antigen-binding fragment thereof does not contain a detectable label. In certain embodiments, the method further comprises using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin) to detect the antibody or antigen-binding fragment thereof.

[0060] In certain embodiments, the second antibody is specific for an antibody of the species (eg, mouse or human) from which the constant region comprised by the antibody or antigen-binding fragment thereof of the invention is derived.

[0061] In certain embodiments, the second antibody is an anti-immunoglobulin (e.g., mouse or human immunoglobulin) antibody, such as an anti-IgG antibody. In certain embodiments, the second antibody is an anti-human IgG antibody. In certain embodiments, the second antibody is an anti-mouse IgG antibody.

[0062] In certain embodiments, the method comprises: (1) contacting the sample with an antibody or antigen-binding fragment thereof of the present invention; (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of herpes virus or cells infected by herpes virus.

[0063] In certain embodiments, the herpesvirus is an alpha herpesvirus. In certain embodiments, the alpha herpesvirus is selected from the genus Herpes Simplex Virus and / or the genus Varicella Herpes Virus. In certain embodiments, the alpha herpesvirus is selected from HSV-1, HSV-2, simian B virus, VZV, PRV or any combination thereof.

[0064] In certain embodiments, described method can be used for diagnostic purposes, for example, can diagnose whether experimenter is infected with herpesvirus according to the existence of herpesvirus in sample or its level, for example the mixed infection of multiple alpha herpesvirus.In such embodiments, described sample can be for example, blood sample (for example, whole blood, blood plasma or serum), excreta, oral cavity or nasal secretion or alveolar lavage fluid from experimenter (for example mammal, preferably people).Therefore, the present invention also provides the method for diagnosing whether experimenter is infected with herpesvirus, it comprises using method as mentioned above to detect the existence of herpesvirus (or its gB protein or the cell infected by herpesvirus) in the sample from experimenter or its level, thereby diagnose whether experimenter is infected with herpesvirus (for example the mixed infection of multiple alpha herpesvirus).

[0065] In certain embodiments, the methods may be used for non-diagnostic purposes, eg, the sample is not a sample from a subject, such as a vaccine sample.

[0066] In another aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, conjugate or kit of the present invention in the preparation of a reagent for detecting the presence or level of herpes virus (or its gB protein, or cells infected by herpes virus) in a sample, and / or for diagnosing whether a subject is infected with herpes virus.

[0067] In certain embodiments, the reagent comprises an antibody or antigen-binding fragment thereof as described above, which optionally comprises a detectable label.

[0068] In certain embodiments, the herpesvirus is an alpha herpesvirus. In certain embodiments, the alpha herpesvirus is selected from the genus Herpes Simplex Virus and / or the genus Varicella Herpes Virus. In certain embodiments, the alpha herpesvirus is selected from HSV-1, HSV-2, simian B virus, VZV, PRV or any combination thereof.

[0069] In certain embodiments, the reagent can be used for diagnostic purposes, such as the sample from a subject's sample, by detecting herpesvirus or its gB protein or the presence or level of cells infected by herpesvirus in the sample, to diagnose whether the subject has been infected with herpesvirus (e.g., mixed infection of multiple alpha herpesviruses) according to the test result. In certain embodiments, the sample is a blood sample (e.g., whole blood, plasma or serum), excreta, oral or nasal secretions or alveolar lavage fluid from a subject (e.g., mammal, preferably human).

[0070] In certain embodiments, the reagents may be used for non-diagnostic purposes, eg, the sample is not a sample from a subject, such as a vaccine sample.

[0071] In certain embodiments, the subject is a mammal, such as a human.

[0072] Definition of terms

[0073] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the procedures of virology, biochemistry, nucleic acid chemistry, immunology, molecular genetics, chemistry, molecular biology, cell culture, microbiology, cell biology, genomics and recombinant DNA used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0074] As used herein, the term "alpha herpesvirus (Alphaherpesvirinae)" belongs to the Herpesviridae family, which is a class of viruses with an envelope and a double-stranded DNA genome, and is divided into four genera: herpes simplex virus, varicella virus, laryngovirus and Malik virus. Alpha herpesvirus generally includes: membrane proteins such as glycoprotein D (gD), glycoprotein H (gH), glycoprotein L (gL), glycoprotein B (gB) and a nucleocapsid that wraps DNA. Currently, 5 known alpha herpesvirus subfamily viruses that can infect humans are HSV-1, HSV-2, simian B virus, VZV and PRV.

[0075] As used herein, the term "gB protein (glycoprotein B)" has a meaning well known to those skilled in the art, and is the most conservative entry glycoprotein in herpesvirus. The gB protein is a type I transmembrane protein, including an extracellular domain, a transmembrane region and a cytoplasmic region. The gB sequences of various herpesviruses are well known to those skilled in the art, and can be found in various public databases. For example, the HSV-1gB gene sequence can be found in GenBank: E03113.1, and an exemplary sequence of its extracellular domain is shown in SEQ ID NO: 13; the HSV-2gB gene sequence can be found in GenBank: MH790666.1, and an exemplary sequence of its extracellular domain is shown in SEQ ID NO: 25; the monkey B virus gB gene sequence can be found in GenBank: LC637779.1, and an exemplary sequence of its extracellular domain is shown in SEQ ID NO: 14; the PRV gB gene sequence can be found in GenBank: ON005001.1, and an exemplary sequence of its extracellular domain is shown in SEQ ID NO: 15; the VZV gB gene sequence can be found in GenBank: 1487662, and an exemplary sequence of its extracellular domain is shown in SEQ ID NO: 16.

[0076] As used herein, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 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 three domains (CH1, CH2 and CH3). 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 domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementarity determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form an antigen binding site.

[0077] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In certain embodiments, the CDRs of the antibodies of the present invention are preferably determined by the IMGT numbering system.

[0078] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0079] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0080] 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 bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (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. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, diabodies, single domain antibodies, chimeric antibodies, nanobodies (technology from Domantis), and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0081] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N to C; and, when the full-length antibody is an IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N to C. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N to C. Two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be from a single species, such as human; or may be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0082] As used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0083] As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0084] As used herein, the term "Fc" means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond. The Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.

[0085] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having an amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0086] As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0087] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also called nanobodies.

[0088] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. In certain embodiments, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0089] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (acceptor antibody). Typically, at least one or two but usually all three (heavy and / or light immunoglobulin chains) of a humanized antibody are replaced by a donor CDR. The immunoglobulin that provides the CDR is called a "donor", and the immunoglobulin that provides the framework is called an "acceptor".

[0090] Chimeric antibodies or humanized antibodies of the present invention can be prepared according to the sequence of monoclonal antibodies produced by immune animals (e.g., mice). DNA encoding heavy and light chains can be obtained from target hybridomas or specific B cells from immune animals and engineered to contain human immunoglobulin sequences using standard molecular biology techniques.

[0091] To prepare chimeric antibodies, methods known in the art can be used to connect the immunoglobulin variable region of an immune animal (e.g., mouse) to a human immunoglobulin constant region. For example, the DNA encoding VH is operably connected to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is generally preferably IgG1 or IgG4 constant region. For example, the DNA encoding VL is operably connected to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0092] To prepare humanized antibodies, the CDR regions of immunized animals (eg, mice) can be transplanted into human framework sequences using methods known in the art (see, e.g., Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0093] Each of the above antibody fragments 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 the antigen.

[0094] Antibody antigen-binding fragments (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

[0095] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0096] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0097] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D In certain embodiments, the dissociation constant can be measured using biomembrane interferometry (BLI) or surface plasmon resonance (SPR).

[0098] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries 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; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can 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 initiation site.

[0099] 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 Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0100] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0101] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. In addition, amino acid residues can also be divided into categories defined by optional physical and functional properties. For example, alcohol-containing residues (S and T), aliphatic residues (I, L, V and M), cycloalkenyl-related residues (F, H, W and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), very small residues (A, G and S), residues involved in turn formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), and flexible residues (Q, T, K, S, G, P, D, E and R). Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0102] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0103] As used herein, the term "subject" refers to a mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from a herpes simplex virus infection or a disease associated with a herpes simplex virus infection, or has the risk of suffering from the above-mentioned disease.

[0104] Advantageous Effects of the Invention

[0105] The present invention provides for the first time a monoclonal antibody with broad-spectrum binding activity to multiple alpha herpes viruses, which can detect and diagnose mixed infections of multiple alpha herpes viruses and has important application value for the detection and diagnosis of herpes virus infections.

[0106] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 The ELISA test results of mAbs 7G4, 12D7 and gB proteins of various alpha herpes viruses are shown.

[0108] Figure 2 The results of immunofluorescence detection of monoclonal antibodies 7G4 and 12D7 binding to gB proteins of various alpha herpes viruses are shown.

[0109] Sequence information

[0110] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0111] Table 1: Description of sequences

[0112]

[0113]

[0114]

[0115] DETAILED DESCRIPTION

[0116] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0117] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE (Animal Cell Culture). CELL CULTURE) (RI Freshney, ed. (1987)).

[0118] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0119] Example 1. Obtaining monoclonal antibody sequences

[0120] 1.1 Preparation of viral antigens:

[0121] For the convenience of writing, the gB protein described in the following examples refers to the extracellular domain of the full-length gB protein. The eukaryotic expression plasmid expressing HSV-1gB protein (SEQ ID NO: 13), HSV-2gB protein (SEQ ID NO: 25), monkey B virus (MBV) gB protein (SEQ ID NO: 14), PRV gB protein (SEQ ID NO: 15), and VZV gB protein (SEQ ID NO: 16) was constructed on the PTT5 vector (obtained from Youbio, VT2202). The above protein was expressed using the 293F eukaryotic expression system. After 7 days of expression, the culture supernatant was collected and affinity purified using an affinity medium. The purified product after elution was collected, and various gB proteins with higher purity were obtained after dialysis and concentration.

[0122] 1.2 Preparation of hybridomas:

[0123] Monoclonal antibody hybridoma cells were obtained using standard in vivo immunization and PEG fusion methods. For detailed methods, see Ed Harlow et al., "Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory 1988. The brief process is as follows:

[0124] (1) Mouse immunization:

[0125] Six-week-old SPF female Balb / C mice were provided by the Experimental Animal Center of Xiamen University.

[0126] The above immunogens were subjected to nucleic acid primary immunization and protein boosting, and mice were immunized in a sequential immunization manner. First, 50 micrograms of eukaryotic expression plasmids expressing HSV-1gB protein, HSV-2gB protein, monkey B virus gB protein, PRV gB protein, and VZV gB protein on the PTT5 vector were sequentially injected with tail vein high pressure, and one immunization cycle was 10 days. Then, 50 micrograms of HSV-1gB protein, PRV gB protein, and VZV gB protein were mixed with incomplete Freund's adjuvant for sequential immunization, and one immunization cycle was 10 days. The mice were immunized with a mixture of 25 micrograms of each of the three proteins, and the spleen of the mice was taken for fusion experiments after 3 days.

[0127] (2) Cell fusion:

[0128] First, the spleen of the mouse was ground to obtain a spleen cell suspension, which was then mixed with mouse myeloma cells SP2 / 0 (obtained from the National Infectious Disease Diagnostic Reagent and Vaccine Engineering Center of Xiamen University) in the logarithmic growth phase, and cell fusion was performed under the action of PEG1450. The fused cells were resuspended in 400 ml of fusion medium and divided into 20 96-well cell culture plates for culture. The fusion medium was RPMI1640 complete screening medium containing HAT and 20% FBS.

[0129] 1.3 Screening of hybridomas:

[0130] After the fusion cells were cultured on a 96-well cell culture plate for 10 days, the cell supernatant was aspirated for ELISA detection, and the detection antigens were HSV-1 gB protein, PRV gB protein, and VZV gB protein. During ELISA detection, the antibodies secreted by the positive wells were required to react specifically with the three gB proteins coated on the polystyrene plate. After three clonings of the positive clones, a monoclonal antibody cell line that could stably secrete antibodies was obtained.

[0131] Hybridoma monoclonal antibody screening results:

[0132] The 7G4 and 12D7 monoclonal antibodies were obtained, which can specifically bind to HSV-1 gB protein, PRV gB protein, and VZV gB protein at the same time.

[0133] 1.4 Hybridoma culture:

[0134] The stable hybridoma monoclonal antibody cell line was first expanded and cultured in a carbon dioxide incubator, transferred from a 96-well to a 24-well, and then transferred to a 50ml cell culture bottle for expansion and culture. The cells in the cell bottle were then collected and injected into the mouse peritoneal cavity, and monoclonal antibody ascites was extracted from the mouse peritoneal cavity after 7-10 days.

[0135] 1.5 Purification of monoclonal antibodies:

[0136] The monoclonal antibody ascites was first precipitated with 50% ammonium sulfate solution, and then the precipitate was dissolved with PBS, and then purified by Protein A column under AKTA system to obtain the purified monoclonal antibody, and the purity of the purified monoclonal antibody was identified by SDS-PAGE.

[0137] The VH / VL sequences of 7G4 are shown in SEQ ID NO:1 / 2, respectively, and the VH / VL sequences of 12D7 are shown in SEQ ID NO:17 / 18, respectively. The CDR sequences of 7G4 and 12D7 were annotated using the IMGT (http: / / www.imgt.org / IMGT_vquest / input) database, and the heavy chain CDR1-3 of 7G4 are shown in SEQ ID NO:3-5, and the light chain CDR1-3 of 7G4 are shown in SEQ ID NO:6-8; the heavy chain CDR1-3 of 12D7 are shown in SEQ ID NO:19-21, and the light chain CDR1-3 of 12D7 are shown in SEQ ID NO:22-24.

[0138] Example 2. Preparation and purification of monoclonal antibodies

[0139] The monoclonal antibody harvested from the hybridoma (the purified monoclonal antibody obtained in Example 1) can be directly used for subsequent activity experiments. Alternatively, the monoclonal antibody can also be prepared by the following genetic engineering recombinant technology.

[0140] The heavy chain variable region of the monoclonal antibody obtained in Example 1 is connected to the heavy chain constant region sequence and constructed into an expression vector to obtain an IgH plasmid expressing the heavy chain of the antibody, and the light chain variable region is connected to the light chain constant region sequence and constructed into an expression vector to obtain an IgK plasmid expressing the light chain of the antibody. The heavy chain constant region sequence can be a heavy chain constant region sequence derived from a mouse or human immunoglobulin (e.g., IgG), such as a human IgG1 heavy chain constant region sequence (SEQ ID NO: 9). The light chain constant region sequence can be a light chain constant region sequence derived from a mouse or human immunoglobulin (e.g., κ or λ), such as a human kappa light chain constant region sequence (SEQ ID NO: 10).

[0141] Tube A: 1mL ExpiCHO TM Expression Medium was used to dilute 12.5 μg of IgH plasmid (SEQ ID NO: 11) and 12.5 μg of IgK plasmid (SEQ ID NO: 12). Tube B: 1 mL of ExpiCHO TM Dilute 80 μL ExpressFectamine in Express Medium TM CHO Transfection Kit transfection reagent. Add tube B to tube A and mix gently. After standing at room temperature for 2 minutes, add 25mL of cells with a density of 6×10 6 / mL, ExpiCHO with cell viability of more than 98% TMCells (obtained from Thermo Scientific) were placed in a cell shaker (125 rpm, 37°C, 8% CO2) for culture. After 18-22 hours of cell culture, 150 μL of enhancer and 4 mL of excipients were added to each 25 mL of CHO cells and transferred to a shaker at 125 rpm, 32°C, 5% CO2 for 8-15 days for mass expression. After expression was completed, the cell supernatant was collected.

[0142] Monoclonal antibodies were purified from CHO cell supernatants using protein A affinity chromatography (GE).

[0143] Example 3. Detection of the binding activity of monoclonal antibodies to alpha herpes virus gB protein

[0144] 3.1 Expression and purification of alpha herpesvirus gB protein

[0145] In order to construct expression plasmids of herpes simplex virus HSV-1 gB protein (SEQ ID NO: 13), HSV-2 gB protein (SEQ ID NO: 25), simian B virus gB protein (SEQ ID NO: 14), PRV gB protein (SEQ ID NO: 15), and VZV gB protein (SEQ ID NO: 16), the nucleotide sequence of amino acids 1-730 was selected with reference to the HSV-1 gB gene sequence (GenBank: E03113.1; amino acids 1-730); the nucleotide sequence of amino acids 1-727 was selected with reference to the HSV-2 gB gene sequence (GenBank: MH790666.1; amino acids 1-904); the nucleotide sequence of amino acids 1-717 was selected with reference to the simian B virus gB gene sequence (GenBank: LC637779.1; amino acids 1-717); the nucleotide sequence of amino acids 1-717 was selected with reference to the PRV gB gene sequence (GenBank: ON005001.1; amino acids 1-752), select the nucleotide sequence of amino acids 1-752; refer to the VZV virus gB gene sequence (GenBank: 1487662; amino acids 1-742), select the nucleotide sequence of amino acids 1-742. The gene fragment was cloned into the mammalian expression vector PTT5 vector for recombinant expression, and the herpes virus gB protein was purified by affinity chromatography.

[0146] 3.2 Preparation of reaction plates

[0147] The herpes virus gB protein obtained above was diluted with 50mM CB buffer (NaHCO3 / Na2CO3 buffer, final concentration of 50mM, pH value of 9.6) at pH9.6, and the final concentration was 1μg / mL. 100μL of coating solution was added to each well of the 96-well ELISA plate, and the coating was carried out at 2-8°C for 16-24 hours and then at 37°C for 2 hours. Wash once with PBST washing solution (20mM PB7.4, 150mMNaCl, 0.1% Tween20). Then 200μL of blocking solution (20mM Na2HPO4 / NaH2PO4 buffer solution with a pH value of 7.4 containing 20% ​​calf serum and 1% casein) was added to each well, placed at 37°C for 2 hours, and the blocking solution was discarded. After drying, it was packed into an aluminum foil bag and stored at 2-8°C for standby use.

[0148] 3.3ELISA

[0149] The monoclonal antibodies 7G4 and 12D7 obtained in Example 2 were diluted to 100 μg / mL with PBS solution, and then diluted 4-fold in 12 gradients. ELISA detection was performed according to the following steps:

[0150] (1) Sample reaction: Take the ELISA plate coated with herpes virus gB protein, add 100 μL of diluted sample to each well, and place it in a 37°C incubator for reaction for 30 minutes.

[0151] (2) Enzyme marker reaction: After completing the sample reaction step, wash the ELISA plate five times with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), add 100 μL HRP-labeled goat anti-mouse IgG (GAM) reaction solution to each well, and place in a 37°C incubator for reaction for 30 minutes.

[0152] (3) Color development reaction: After completing the enzyme labeling reaction step, the ELISA plate was washed five times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), and 50 μL of TMB color developer (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to each well and placed in a 37°C incubator for reaction for 15 minutes.

[0153] (4) Termination of reaction and measurement of readings: After the color development step is completed, 50 μL of stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) is added to each well of the ELISA plate after the reaction, and the OD450 / 630 value of each well is measured on an ELISA reader.

[0154] Determination of the reactivity of monoclonal antibodies 7G4 and 12D7 with herpes virus gB protein: Determine based on the reading after the reaction. If the detection value / background value is greater than 5, it is determined to be positive.

[0155] (5) Result analysis: Figure 1 As shown, monoclonal antibodies 7G4 and 12D7 have strong binding activity to herpes virus HSV-1 gB protein, simian B virus gB protein, PRV gB protein, and VZV gB protein.

[0156] 3.4 Immunofluorescence detection

[0157] Cells containing HSV-1, HSV-2, PRV, and VZV were obtained respectively, wherein: HSV-1 strain 17 (isolated from clinical samples) and PRV Min A strain virus (isolated from sick pigs) were infected with MDCK (canine kidney cells, obtained from the University of Hong Kong) at a dose of MOI = 0.1, and cultured in a carbon dioxide incubator at 37°C for 24 hours; VZV-Oka virus (derived from attenuated vaccine) was used to infect ARPE-19 (human retinal cells, obtained from Rutgers University New Jersey Medical School) cells at a dose of MOI = 0.1, and cultured in a carbon dioxide incubator at 37°C for 48 hours; and the expression plasmid expressing HSV-2gB was transfected into MDCK cells using a commercial Lipo3000 transfection reagent, and cultured in a carbon dioxide incubator at 37°C for 48 hours. The culture supernatant was removed, the cells were washed 3 times with PBS (phosphate) buffer, fixed with 5% paraformaldehyde solution for 15 minutes, washed 3 times with PBS solution, then permeabilized with PBST solution for 15 minutes, washed 3 times with PBS solution, and blocked with 200 μl of goat serum for 2 hours. The blocking solution was removed, 7G4 and 12D7 antibodies of appropriate concentrations were added, incubated at 4°C for two hours, washed 5 times with PBS solution, and polyclonal antibodies of goat anti-mouse antibodies labeled with FITC (fluorescein isothiocyanate) of appropriate concentrations were added, incubated at 4°C for two hours, washed 5 times with PBS solution, and then the virus-infected cells were observed for fluorescence positivity using a high-content imaging system.

[0158] The experimental results are as follows Figure 2 As shown, 7G4 and 12D7 can specifically bind to herpes virus HSV-1 gB protein, HSV-2 gB protein, PRV gB protein, and VZV gB protein. The above results show that 7G4 and 12D7 have broad-spectrum binding activity to alpha herpes viruses and have important application value for the detection and diagnosis of mixed infection of multiple alpha herpes viruses.

[0159] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to herpes simplex virus gB protein, the antibody or the antigen-binding fragment thereof comprising: (a) a heavy chain variable region (VH) comprising the following three complementary determining regions (CDRs): VH CDR1 of SEQ ID NO: 3, VH CDR2 of SEQ ID NO: 4, and VH CDR3 of SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three complementary determining regions (CDRs): VL CDR1 of SEQ ID NO: 6, VL CDR2 of SEQ ID NO: 7, and VL CDR3 of SEQ ID NO: 8; or (b) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VHCDR1 of SEQ ID NO: 19, VH CDR2 of SEQ ID NO: 20, and VH CDR3 of SEQ ID NO: 21; and / or, a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs): VL CDR1 of SEQ ID NO: 22, VL CDR2 of SEQ ID NO: 23, and VL CDR3 of SEQ ID NO:

24.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 2 or a variant thereof; or, (b) a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 17 or a variant thereof; and / or a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 18 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO: 2; Preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO:17 and a VL comprising the sequence shown in SEQ ID NO:

18.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a constant region derived from an immunoglobulin; Preferably, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from a mouse or human immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3 or IgG4), and the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from a mouse or human immunoglobulin (e.g., κ or λ).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or antigen-binding fragment thereof is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, single domain antibody (sdAb), chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The herpes virus is an alpha herpes virus; Preferably, the alpha herpesvirus is selected from the genus Herpes simplex virus and / or the genus Varicella herpes virus; Preferably, the alpha herpes virus is selected from HSV-1, HSV-2, simian B virus, PRV, VZV or any combination thereof.

6. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the heavy chain variable region and / or light chain variable region thereof; Preferably, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

7. A vector comprising the nucleic acid molecule according to claim 6; Preferably, the vector is a cloning vector or an expression vector; Preferably, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors.

8. A host cell comprising the nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising culturing the host cell according to claim 8 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

10. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and a detectable label linked to the antibody or antigen-binding fragment thereof; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

11. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the conjugate according to claim 10; Preferably, the kit comprises the conjugate of claim 10; Preferably, the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

12. A method for detecting the presence or level of herpes virus in a sample, comprising using the antibody or antigen-binding fragment thereof of any one of claims 1 to 5, the conjugate of claim 10, or the kit of claim 11; Preferably, the method is an immunological assay, such as an enzyme immunoassay (eg ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay or a radioimmunoassay; Preferably, the method comprises using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin); Preferably, the herpes virus is an alphaherpes virus; Preferably, the alpha herpesvirus is selected from the genus Herpes simplex virus and / or the genus Varicella herpes virus; Preferably, the alpha herpes virus is selected from HSV-1, HSV-2, simian B virus, PRV, VZV or any combination thereof; Preferably, the method is performed in vitro; Preferably, the method is used for non-diagnostic purposes.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the conjugate according to claim 10, or the kit according to claim 11 in the preparation of a reagent for detecting the presence or level of herpes virus in a sample, and / or for diagnosing whether a subject is infected with herpes virus; Preferably, the herpes virus is an alphaherpes virus; Preferably, the alpha herpesvirus is selected from the genus Herpes simplex virus and / or the genus Varicella herpes virus; Preferably, the alpha herpes virus is selected from HSV-1, HSV-2, simian B virus, PRV, VZV or any combination thereof; Preferably, the sample is a blood sample (eg, whole blood, plasma or serum), feces, oral or nasal secretions, or alveolar lavage fluid from a subject (eg, a mammal, preferably a human).

Images