DIAGNOSIS AND TREATMENT FOR CHRONIC INFLAMMATION AND VIRUS INFECTION
20240043511 ยท 2024-02-08
Assignee
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
G01N33/6863
PHYSICS
C07K2299/00
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present application provides a diagnosis and treatment method for chronic inflammation. The technical solution provided by the present application is an application of a reagent in preparing a product for preventing and/or treating chronic inflammation diseases: the reagent is a substance for inhibiting the activity of an abnormal content of IFP35 and/or NMI which is secreted outside the cell as an inflammatory factor. Experiments prove that using antibodies and the like to inhibit the activity of an abnormal content of IFP35 and/or NMI which is secreted outside the cell as an inflammatory factor can effectively treat chronic inflammation diseases. In the present application, IFP35 and/or NMI are also used as a target spot, providing a diagnosis/auxiliary diagnosis and treatment method and tool for infection of viruses, particularly novel coronavirus-19 (COVID-19).
Claims
1-49. (canceled)
50. A method for treating and/or alleviating a chronic inflammatory disease, or an inflammatory response in an individual infected with a virus, comprising administering to the individual a therapeutically effective amount of an antibody or an antigen-binding fragment thereof which specifically binds to IFP35 and/or NMI.
51. The method according to claim 50, wherein the antibody is the antibody having a light chain variable region and a heavy chain variable region, and the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are set forth in SEQ ID NOs: 13, 14 and 15 respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region are set forth in SEQ ID NOs: 16, 17 and 18, respectively; the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 19, 14 and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 18, respectively; the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 20, respectively; the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 21, respectively; the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 22, respectively; or the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 23, respectively.
52. The method according to claim 50, wherein the sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 9, and the sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 10.
53. The method according to claim 50, wherein the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 25, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 24; the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 1, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 3; the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 5, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 7; or the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 11, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 12.
54. The method according to claim 50, wherein the chronic inflammatory disease is selected from the group consisting of multiple sclerosis, arthritis, rheumatoid arthritis, psoriasis, various enteritis such as inflammatory bowel disease (IBD), asthma, chronic obstructive pulmonary disease and systemic lupus erythematosus, chronic hepatitis, chronic nephritis, chronic pancreatitis, encephalitis, malignant tumors, leukemia, Alzheimer's disease, Parkinson's syndrome and the like.
55. The method according to claim 54, wherein the abnormal amount of the secreted inflammatory factor IFP35/NMI is involved in the chronic inflammatory disease comprising rheumatoid arthritis (RA), osteoarthritis (OA), multiple sclerosis (MS), atherosclerosis, myocardial infarction, chronic obstructive pulmonary disease (COPD), chronic nephritis, chronic hepatitis, chronic pancreatitis, type 2 diabetes, systemic lupus erythematosus (SLE), Alzheimer's disease, Parkinson's disease (PD) malignant tumors, asthma, allergic diseases, cardiovascular diseases, musculoskeletal diseases, inflammatory bowel disease (IBD), obesity and diabetes, retinal inflammatory disease (AMD), periodontitis, uveitis and the like.
56. The method according to claim 50, wherein the individual infected with a virus is a severe or critical diseased individual.
57. The method according to claim 50, wherein the virus is a Coronaviridae virus, such as novel coronavirus (COVID-19), SARS virus, MERS virus; or a Orthomyxoviridae virus, for example Influenza Virus, such as Influenza A Virus.
58. The method according to claim 50, wherein the individual is a mammal, such as a human.
59. A method for diagnosing a chronic inflammatory disease, or an inflammatory response in an individual infected with a virus, comprising determining an amount of interferon-induced protein 35 kD (IFP35) and/or N-Myc interacting protein (NMI) in a biological sample obtained from the individual.
60. The method according to claim 59, wherein the chronic inflammatory disease is related to increased inflammatory response caused by abnormal secretion of IFP35 and/or NMI into blood or body fluids, and comprises arthritis, rheumatoid arthritis, psoriasis, various enteritis such as inflammatory bowel disease (IBD), multiple sclerosis, asthma, chronic obstructive pulmonary disease, systemic lupus erythematosus, chronic hepatitis, chronic nephritis, chronic pancreatitis, encephalitis, malignant tumors, leukemia, Alzheimer's disease, Parkinson's syndrome, allergic diseases, cardiovascular diseases, musculoskeletal diseases, inflammatory bowel disease, obesity and diabetes, retinal inflammatory disease, periodontitis, uveitis and the like; or the chronic inflammatory disease is related to increased inflammatory response caused by abnormal secretion of IFP35 and/or NMI into blood or body fluids, and comprises arthritis, rheumatoid arthritis, psoriasis, various enteritis such as inflammatory bowel disease (IBD), multiple sclerosis, asthma, chronic obstructive pulmonary disease, systemic lupus erythematosus, chronic hepatitis, chronic nephritis, chronic pancreatitis, encephalitis, malignant tumors, leukemia, Alzheimer's disease, Parkinson's syndrome, allergic diseases, inflammatory bowel disease and the like, and wherein the chronic inflammation disease has been repeatedly proved to be related to a variety of inflammatory factors.
61. The method according to claim 59, wherein the diagnosis is early diagnosis, diagnosis of a condition and prognosis judgment.
62. The method according to claim 59, wherein the individual infected with a virus is a severe or critical diseased individual.
63. The method according to claim 59, wherein the virus is a Coronaviridae virus, such as novel coronavirus (COVID-19), SARS virus, MERS virus; or a Orthomyxoviridae virus, for example Influenza Virus, such as Influenza A Virus.
64. The method according to claim 59, wherein the individual is a mammal, such as a human.
65. The method according to claim 59, wherein determining the amount of IFP35 and/or NMI in the biological sample is determining an amount of IFP35 and/or NMI protein in the biological sample, or determining an expression level of IFP35 and/or NMI nucleic acid in the biological sample, such as an amount of mRNA.
66. The method according to claim 59, the biological sample is blood, plasma, serum, cerebrospinal fluid, or alveolar lavage fluid.
67. An antibody or an antigen-binding fragment thereof, wherein the antibody having a light chain variable region and a heavy chain variable region, and the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 19, 14 and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 18, respectively; the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 20, respectively; the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 21, respectively; the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 22, respectively; or the sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody are set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody are set forth in SEQ ID NOs: 16, 17 and 23, respectively.
68. The antibody or the antigen-binding fragment thereof according to claim 67, wherein the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 25, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 24; or the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 1, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 3; or the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 5, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 7; or the sequence of the heavy chain constant region of the antibody is set forth in SEQ ID NO: 11, and the sequence of the light chain constant region of the antibody is set forth in SEQ ID NO: 12.
69. A pharmaceutical composition for treating or alleviating a chronic inflammatory disease, or an inflammatory response in an individual infected with a virus, comprising a therapeutically effective amount of the antibody or the antigen-binding fragment thereof according to claim 67, and a pharmaceutically acceptable carrier.
Description
DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0229] The following examples are provided only to illustrate some embodiments of the present application, without purpose or nature of any limitations.
Example 1: Treatment of MS in Mice with an Antibody
[0230] A mouse MS model was established, and then treated with an antibody for IFP35. The result is shown in
Example 2: Treatment of MS in Mice with Nucleic Acid Drugs
[0231] The NMI gene was knocked out in mice. The symptoms of MS in mice with the knockout of NMI gene were then observed. The results are shown in
Example 3: Detection of MS in Mice with IFP35/NMI as Markers in the Serum or Body Fluid (Such as Cerebrospinal Fluid and the Like)
[0232] Cell experiments showed that compared with normal cells, the expression level of IFP35/NMI in the macrophages induced by LPS, MOG and H37Ra (similar to MS animal models) increased significantly, and IFP35/NMI was secreted to the outside the cell. Therefore, the increased amount of IFP35/NMI can be detected in the serum. At the same time, it was found that the expression levels of other inflammatory cytokines and their amounts in serum were also increased, for example the mRNA expression levels of the markers of M1 type microglia TNF, iNOS, and IL10 were increased under the stimulation of LPS and NMI along with iNOS, HMGB1, TNF, iNOS and IL1, as shown in
[0233] MS animal model experiments showed (see experimental methods) that compared with normal mice, the expression level of IFP35/NMI in the MS mouse model was significantly increased, and IFP35/NMI was secreted into the serum. The high expression level and release of IFP35/NMI, as an inflammatory factor, were consistent with those of other MS characteristic inflammatory factors such as TNF, iNOS, and IL10 in the MS mouse model. It indicates that IFP35/NMI may be used as a characteristic biomarker and detection indicator of MS in serum or body fluid, as shown in
[0234] Therefore, it is possible to detect whether the expression level of IFP35/NMI in the spinal cord tissue of mice is significantly increased, or whether IFP35/NMI is secreted into the blood or body fluids (such as cerebrospinal fluid) of the mice and the correlation of the increased amount of IFP35/NMI secreted into the blood or body fluids to assist in diagnosing whether the mice may have MS and determining the inflammatory factors that cause the disease. If the expression level of IFP35/NMI in the tissue of mice is abnormally increased, IFP35/NMI is abnormally secreted into the blood or body fluid of mice (such as cerebrospinal fluid), or the increased expression level of IFP35/NMI is consistent with the expression levels of other inflammatory factors (such as interferon, TNF, IL1, IL6 and the like) and their secretion into blood in mice, which causes obvious external symptoms such as changes in the gait and paralysis of the limbs in mice, the mice can be substantially diagnosed with MS.
Experimental Methods
BMDM Culture:
[0235] The mice were sacrificed by cervical dislocation. Two hind legs of the mice were taken and soaked in 70% ethanol for 1 minute, and the muscles on the hind leg bones were removed as much as possible. The PBS containing penicillin and streptomycin was sucked into the syringe to flush the bone marrow cavity. After centrifuging at 400 g for 10 minutes, the supernatant was removed. 1 ml red blood cell lysis buffer was used to lyse the red blood cells for 30 seconds, and then 10 ml PBS was added to neutralize the red blood cell lysis buffer. After centrifuging at 400 g for 10 minutes, the cells were cultured in an incubator at 37 C., 5% CO.sub.2 with DMEM (Gibico) complete culture medium containing 10% FBS (Gibco), 1% penicillin and streptomycin, 1% L-glutamine and MCSF (peprotech) at a final concentration of 20 ng/ml.
Raw264.7 Cell Culture:
[0236] The cells were cultured in an incubator at 37 C., 5% CO.sub.2 with DMEM (Gibico) complete culture medium containing 10% FBS (Gibco), 1% penicillin and streptomycin and 1% L-glutamine.
Thp1 Cell Culture:
[0237] The cells were cultured in an incubator at 37 C., 5% CO.sub.2 with 1640 (Gibico) complete culture medium containing 10% FBS (Gibco), 1% penicillin and streptomycin and 1% L-glutamine.
BV2 Cell Culture:
[0238] The cells were cultured in an incubator at 37 C., 5% CO.sub.2 with DMEM (Gibico) complete culture medium containing 10% FBS (Gibco), 1% penicillin and streptomycin and 1% L-glutamine.
Stimulation of Cells with MOG and H37Ra:
[0239] MOG and H37Ra were added to the above-mentioned cells at a final concentration of 100 ng/ml to stimulate for 8h.
Stimulation of BV2 Cells with mNMI:
[0240] The mouse recombinant protein mNMI was expressed and used to stimulate mouse primary microglia in vitro. Cells were adjusted to the concentration of 210.sup.6 and incubated with 5 g/ml mNMI for 6h, and then the polarization markers of macrophages were detected by QPCR.
Total RNA Extraction from Cells:
[0241] RNA extraction kit (Cat No. CW 0597, CoWin Biosciences) was used for total RNA extraction for BMDM cells. Trizol (invitrogen) was used for total RNA extraction for BV2 cells.
Reverse Transcription of mRNA into cDNA:
[0242] PrimeScript II 1st Strand cDNA Synthesis Kit (Cat.No.6210A) and SYBR Premix Ex Tag (Tli RNaseH Plus), ROX plus Q-PCR kit (Cat. NO. RR42LR) were all purchased from TAKARA Bio company.
Establishment of MS Mouse Model (EAE):
[0243] The mice selected for the experiments were female C57BL/6 mice aged 8-12 weeks, which were purchased from Vital River Laboratory Animal Technology Co., Ltd. The oligodendrocyte protein MOG35-55 and Freund's complete adjuvant CFA were used for emulsification. Each mouse was injected subcutaneously with 0.2 mg MOG35-55 and 0.2 mg CFA, combined with pertussis toxin PTX. Each mouse was injected intraperitoneally with 500 ng PTX, and the injection was repeated once after 48h. The sham treatment group used the same treatment except that PBS was given instead of MOG35-55. The disease and life status of the mice were observed and recorded every day. The mice were scored after the onset of the disease, and the scoring standards were as follows: 0, no clinical symptoms; 1. paralysis of the tail of the mouse; 2. paralysis of one hind limb or weakness of both hind limbs of the mouse; 3. paralysis of both hind limbs of the mouse; 4. paralysis of both hind limbs and the affected forelimbs of the mouse; 5. the mouse is dying. The diseased mice were scored and evaluated according to the scoring standards.
Extraction of Whole Proteins from Mouse Spinal Cord Tissue:
[0244] 100 mg of perfused mouse spinal cord tissue was obtained, and lysed for 30 minutes on ice after adding RIPA (Proteinase inhibitor cocktail, Roche). The lysate was centrifuged at 12,000 rpm and 4 C. for 10 minutes, and 5SDS-PAGE loading buffer was added to the pellet and kept at 95 C. for 5 minutes.
Detection of Secreted NMI/IFP35 in the Serum of Mouse Infected with Influenza Virus
[0245] C57BL/6 WT, IFI35.sup./ and NMI.sup./ mice were infected with A/PR8 strain at a dose of 300 pfu as the challenge experimental groups, and wild-type C57BL/6 mice were inoculated with the same dose of PBS as a negative control group. Whether the protein levels of IFP35 and NMI in the serum of mice change was detected on day 3 post the virus infection, and the amounts of TNF and IL6 in the serum were detected.
Preparation of a Neutralizing Antibody Against IFP35:
[0246] Mice were injected intraperitoneally with monoclonal hybridoma cells for IFP35. The mouse ascites was collected 7-10 days after injection.
[0247] The antibodies in the ascites were purified by Protein G Agrose beads (GE): [0248] (1) adding 1 ml Protein G Agrose beads slurry to the packed Column for protein purification; [0249] (2) washing the matrix in the purification column with 10 ml phosphate buffer; [0250] (3) adding 1 ml ascites to 5 ml phosphate buffer, and then adding the mixture to the purification column, which was incubated at 4 C. for 2 hours; [0251] (4) washing: washing the column with 20 ml phosphate buffer; [0252] (5) eluting: eluting the antibody with an appropriate amount of 0.1M Glycine (pH=2.5-3.0) buffer, and the elution solution was neutralized with 1M Tris-HCl buffer (pH=10.0) which was added in advance to the elution tube;
[0253] The antibody was concentrated with an ultrafiltration tube, and the concentration of the antibody was determined.
Treatment of Mice with MS Using an Antibody Against IFP35:
[0254] The mouse EAE models were established and divided into the treatment group with the antibody against IFP35 and the control group with an IgG antibody. The antibodies were administered to the mice from 12 days after modeling. Each mouse was injected intravenously with 100 g (5 mg/kg) antibody every day for 10 consecutive days. Clinical symptoms of the two groups of mice were compared.
ELISA Assay:
[0255] (1) The plates were taken out and returned to room temperature. The diluted standards and samples were added to the plates with 100 ml per well and incubated for 2 h at room temperature. Standards and samples were diluted with 1% BSA in PB ST (0.05% Tween-20). [0256] (2) The wells were washed with PBST (0.05% Tween-20) for 3 times and deionized water for 2 times. [0257] (3) The detection antibodies were added to the wells with 100 ml per well and incubated for 2 h at room temperature. The detection antibodies were diluted with 1% BSA in PBST (0.05% Tween-20). [0258] (4) The substrate was added to the wells for color development, with 100111 TMB per well. The plates were shaken on a shaker for decolorizing for 10-20 minutes in the dark. [0259] (5) 50 ml 2 mol/L H2504 was added to each well to stop the reaction.
[0260] The OD values were measured at 450 nm using a microplate reader.
[0261] A method for obtaining the gene knock-out mice: 8-12 weeks old C57BL/6 mice were used. All mice used were sacrificed by cervical dislocation. The wild-type C57BL/6 mice (000664) were purchased from Vital River Laboratory Animal Technology Co., Ltd. CRISPR-Cas9 technology was used to generate NMI and Ifp35 gene knock-out mice. The fertilized eggs were collected from the fallopian tubes of the mice having superovulation. The female C57BL/6 mice were mated with the male C57BL/6 mice. Cas9 mRNA (150 ng ml-1) was mixed with sgRNA (100 ng ml-1) produced by transcription and the mixture was microinjected into the cells. The cytoplasm of the fertilized egg has a recognized pronucleus in M2 medium (Sigma, M7167-100ML). The sgRNA sequence of NMI is 5-AAAACAAAGAACTAGACGAGG-3 (SEQ ID NO:26), and the sgRNA sequence of IFP35 is 5-CAGCTCAAAAGGGAGCGCACAGG-3 (SEQ ID NO:27). The frameshift mutation of NMI and Ifp35 genes produced by CRISPR technology resulted in the failure to produce NMI and IFP35 proteins. A corresponding sgRNA was injected into about 100-250 fertilized eggs, and then the fertilized eggs were transferred into the uteruses of surrogate ICR female mice to obtain F1 generation mice.
Results:
[0262] 1. MOG and H37Ra Induced NMI Release from Mouse Macrophages.
[0263] A method involved in
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2. LPS Induced the Release of NMI from Microglia BV2.
[0265] A method involved in
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3. mNMI Protein Induced M1 Polarization of Mouse Microglia.
[0267] A method involved in
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4. The Secretory Expression of NMI in Mice at Different Times after EAE Modeling.
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5. The Expressions of NMI and Other Inflammatory Factors in Mouse Spinal Cord Tissue after EAE Modeling.
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[0271] A summary of the results shown in
[0272] This experiment can prove that in the animal model with MS, the expression level of NMI increased and NMI was released into the blood, but it cannot prove that NMI promotes the release of other inflammatory factors, as NMI and other inflammatory factors iNOS, COX2 and HMGB1 are detected together. There is no sequential and causal relationship between the expressions of NMI and other inflammatory factors.
6. The Knockout of NMI Gene Ameliorated the Clinical Symptoms of MS in Mice.
[0273] A method involved in
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7. An Antibody for IFP35 Alleviated the Symptoms of MS in Mice.
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[0276] The corresponding antibody in this experiment is 35NIDmAb with the light chain variable region being set forth in SEQ ID NO: 10, and the heavy chain variable region being set forth in SEQ ID NO: 9.
[0277] The heavy chain variable region comprises CDR1, CDR2 and CDR3, with the sequence of CDR1 consisting of amino acid residues at positions 25-32 of SEQ ID NO: 9 (GYTFTNYG), the sequence of CDR2 consisting of amino acid residues at positions 50-57 of SEQ ID NO: 9 (INTYTGEP), and the sequence of CDR3 consisting of amino acid residues at positions 98-106 of SEQ ID NO: 9 (YGYSWAMDY).
[0278] The light chain variable region comprises CDR1, CDR2 and CDR3, with the sequence of CDR1 consisting of amino acid residues at positions 26-31 of SEQ ID NO: 10 (SSSVSY ), the sequence of CDR2 consisting of amino acid residues at positions 49-51 of SEQ ID NO: 10 (DTS), and the sequence of CDR3 consisting of amino acid residues at positions 90-96 of SEQ ID NO: 10 (WSSNPPI). The amino acid sequences of CDRs are numbered according to Kabat system.
[0279] In MS animal models and the experiments of inducing macrophages by MS-inducing reagents such as MOG and the like, it was found that IFP35/NMI was released into the serum. Therefore, this application is to study the relationship between the released IFP35/NMI and a disease. As IFP35/NMI has been previously proved by the present inventors that once being released into the serum, it will play a role of DAMPs. Therefore, this study actually refers to the role of IFP35/NMI released into the serum in the disease, that is, the function of DAMP.
8. The Knockout of NMI Gene Reduced the Infiltration of Spinal Cord Leukocytes and the Inflammation in MS Mice
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9. Comparison of NMI and PCT in Serum of Mice with LPS-Induced Inflammation
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10. The Secretion of NMI/IFP35 was Detected in Mouse Serum after the Mice were Infected with Influenza A/Puerto Rico/8/1934 (PR8) Virus Strain.
[0282] As a control, the present inventors also compared the secretion of IFP35 and NMI in the case of virus infection. The research method comprises: infecting C57BL/6 WT, and NMI.sup./ mice with PR8 strain at a dose of 300 pfu as the challenge experimental groups, and inoculating the wild-type C57BL/6 mice with the same dose of PBS as the negative control group. As shown in panels A and B of
Example 4: Improvement of an Antibody
I. The Humanization of an Antibody:
1. Experimental Method
[0283] Mutations were introduced into the DNA sequence of specific amino acid residues of the mouse antibody by PCR site-directed mutagenesis to modify some amino acids of the mouse antibody and transform it into a humanized antibody, realizing the humanization of the antibody. Then the engineered antibody gene was cloned into the corresponding antibody expression vector, such as pCDNA3.1, pCDNA3.4 and so on. The expression vector was transformed into human kidney cell line eExpi293F or HEK293T cells for expression, and then the expressed antibody secreted to the outside of the cells was purified. The purified engineered antibody and the antigen IFP35 were detected for their binding affinity. The equipment used is BiACo or ITC and so on. The ELISA method can also be used to detect the binding ability of the humanized antibody to the antigen. The antigen is the human IFP35 protein.
[0284] The sequences of the mouse antibody (35NIDmAb) before and after engineering are as follows:
[0285] The sequence of the light chain constant region of AE001VL (the mouse antibody before engineering (35NIDmAb) is set forth in SEQ ID NO: 24; the sequences of the light chain constant regions of the humanized mouse antibodies AE001L1, AE001L2 and AE001L3 after engineering are respectively set forth in SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO:12.
[0286] The sequence of the heavy chain constant region of AE001VH (the mouse antibody before engineering (35NIDmAb) is set forth in SEQ ID NO: 25; the sequences of the heavy chain constant regions of the humanized mouse antibodies AE001H1, AE001H2 and AE001H3 after engineering are respectively set forth in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO:11.
[0287] The experimental results are shown in
II. The Engineering of the Antibody Affinity Maturation
1. Experimental Method
[0288] The experiment adopted a universal method for mutant plasmid construction and a method for protein antibody expression. Firstly, random mutations were introduced into the DNA sequence of the CDR regions of a humanized antibody AE001-H3+L3 by PCR random mutation method, thereby changing the CDR sequences of the humanized antibody. Then the engineered antibody sequence was cloned into the corresponding antibody expression vector, such as pCDNA3.1, pCDNA3.4 and so on. A random mutant antibody library was established. The library plasmids were transferred into human kidney cell line 293T or Expi293F cells for expression, and then the expressed antibodies secreted to the outside of the cells were purified. The purified engineered antibody and the in vitro purified antigen IFP35 or NMI or IFP35/NMI complex were detected for their binding affinity. The equipment used is BiACo or ITC and so on. The ELISA method can also be used to detect the ability of the antibody to bind to the antigen after the CDR regions has been modified.
2. Results
[0289] 1) The preliminary screening results of detecting affinity of a random mutant antibody by ELISA method:
TABLE-US-00005 TABLE 1 the preliminary screening of the random mutant antibody AE001H3 + L3 Antigen AE001-bio(2 g/ml), room temperature, 1 h Block 2% MPBS, room temperature, 2 h Sample The cell samples expressing the random mutation antibody AE001- H3 + L3 1 2 3 4 5 6 7 8 9 10 11 12 A 0.2488 0.1232 0.1617 0.2786 0.1487 0.2955 0.1672 0.1193 0.2639 0.2213 0.2195 0.2451 B 0.1684 0.2336 0.178 0.2487 0.3215 0.252 0.2602 0.1315 0.2295 0.258 0.3118 0.2648 C 0.2473 0.198 0.323 0.2212 0.2003 0.2105 0.1746 0.1714 0.1745 0.2208 0.257 0.1827 D 0.2656 0.2543 0.2113 0.2343 0.1948 0.2887 0.1469 0.0806 0.1886 0.2384 0.2397 0.271 E 0.2899 0.1644 0.23 0.4359 0.2324 0.43 0.0924 0.2081 0.2477 0.2833 0.2262 0.2488 F 0.2702 0.2388 0.2431 0.1882 0.18 0.1914 0.0817 0.1235 0.1961 0.1818 0.1689 0.1767 G 0.3287 0.2274 0.2396 0.3342 0.2444 0.2955 0.2068 0.1165 0.0941 0.116 0.1931 0.1989 H 0.2247 0.2264 0.2279 0.2273 0.1784 0.2193 0.153 0.1673 0.1948 0.225 0.1815 0.1717 The Anti-human Kappa + Lambda chain HRP(1:4000), room temperature, 1 h secondary antibody color TMB for color development, room temperature, 10 min development termination 2M hydrochloric acid
[0290] Five antibodies with better binding ability (antibodies underlined above) were selected based on the above results, which were numbered as AE001-5, AE001-6, AE001-7, AE001-8, AE001-9, respectively. The details of the antibodies are as follows. [0291] 2) The pairing of the heavy and light chains of the mutant antibodies in the preliminary screening:
TABLE-US-00006 The numberings of The pairings of The subtype of antibodies antibodies antibodies AE001-5 AE001-5Hc + Lc Human IgG1 AE001-6 AE001-6Hc + Lc Human IgG1 AE001-7 AE001-6Hc + 7Lc Human IgG1 AE001-8 AE001-6Hc + 8Lc Human IgG1 AE001-9 AE001-6Hc + 9Lc Human IgG1 [0292] 3) The antibody expression and purification are shown in
TABLE-US-00007 Loading Sample ID KD (M) kon(1/Ms) kdis(1/s) Full R{circumflex over ()}2 AE001-H3 + L3 1.39E08 1.45E+05 2.01E03 0.9953 AE001-6HC + LC 3.35E09 1.52E+05 5.08E04 0.9982 AE001-6HC + 8LC 2.99E09 1.47E+05 4.40E04 0.9985 AE001-6HC + 9LC 3.04E09 1.49E+05 4.54E04 0.9986 AE001-6HC + 7LC 3.46E09 1.51E+05 5.24E04 0.9980 AE001-5HC + LC 5.47E09 1.69E+05 9.22E04 0.9964
[0295] Conclusion: The 5 preferred antibodies (numbered as 5-9) obtained have different degrees of affinity improvement. The results of affinity kinetics determination (fortebio) shows that each antibody has a 2.5-4.6-fold increase in affinity; the increase in affinity is mainly due to the great improvement of K.sub.off, and the clone affinity of AE001-6HC+8LC (the antibody AE001-8) shows the best improvement.
[0296] Changes in the Heavy Chain Sequences:
TABLE-US-00008 The CDR sequences of the heavy chain The original sequence (SEQ ID CDR1(25-32) CDR2(50-57) CDR3(98-106) NO: 9) GYTFTNYG INTYTGEP YGYSWAMDY AE001- AE001-5 GYTFPNYG unchanged unchanged 5Hc + Lc AE001- AE001-6 unchanged unchanged unchanged 6Hc + Lc AE001- AE001-7 unchanged unchanged unchanged 6Hc + 7Lc AE001- AE001-8 unchanged unchanged unchanged 6Hc + 8Lc AE001- AE001-9 unchanged unchanged unchanged 6Hc + 9Lc
[0297] Changes in the Light Chain Sequences:
TABLE-US-00009 The CDR sequences of the light chain The original sequence (SEQ ID CDR1(26-31) CDR2(49-51) CDR3(90-96) NO: 10) SSSVSY DTS WSSNPPI AE001- AE001-5 unchanged unchanged unchanged 5Hc + Lc AE001- AE001-6 unchanged unchanged WSPYPPI 6Hc + Lc AE001- AE001-7 unchanged unchanged WSSNSWS 6Hc + 7Lc AE001- AE001-8 unchanged unchanged WSPLPPI 6Hc + 8Lc AE001- AE001-9 unchanged unchanged WSPRPPI 6Hc + 9Lc Note: the cyan-labeled residues are antigen-binding residues.
CONCLUSION
[0298] The antibodies with optimized antigen-antibody binding capacity and the better neutralizing activity can be obtained by modifying some amino acid residues of the CDR sequences of the originally screened antibodies. It can be seen from the above tables that some antibodies that can bind to the antigen IFP35 were screened and obtained by modifying a part of CDR sequences of the original antibodies. Modifying some amino acid sequences of the above mentioned CDRs will be useful to obtain antibodies with better activity. Modifying a part of CDR sequences is an important method commonly used internationally to obtain more optimized antibodies. Therefore, an antibody that comprises the CDR sequences having 50% or more identity (the amino acids are at the same CDR positions and have the same sequence numberings) to the CDR sequences of the antibodies disclosed in the present application and binds to IFP35 shall fall into the scope of the present application.
Example 5: The Fine Three-Dimensional Crystal Structure of the Complex of the Neutralizing Antibody and the Antigen IFP35 Reveals the Key Residues for the Antibody to Recognize the Antigen and the Characteristics of the Amino Acid Residues of the IFP35 Antigenic Epitope
[0299] The mice were immunized using the NID domain of IFP35 (a fragment consisting of amino acids at positions 124-220) as an immunogen. A monoclonal antibody against IFP35 was screened and obtained from the mouse spleen, which was named as 35NIDmAb.
[0300] The monoclonal antibody 35NIDmAb is a monoclonal antibody targeting IFP35 NID (a fragment consisting of amino acids at positions 124-220) with neutralizing activity. The 35NIDmAb targeting IFP35 NID binds strongly to IFP35 NID (Kd=28-10.sup.9 M). In vitro purification experiments show that after 35NIDmAb and IFP35 NID were mixed and purified by gel filtration chromatography, they still bind to each other. These results indicate that the 35NIDmAb targeting IFP35 NID can bind to IFP35 NID strongly, and has a neutralizing activity. The aforementioned experiments have proved that the monoclonal antibody can protect mice and ameliorate multiple sclerosis (MS) symptoms of mice.
[0301] In order to clarify the structural basis for the neutralizing activity of 35NIDmAb on IFP35 NID, based on the high affinity binding of 35NIDmAb and IFP35 NID, the present inventors analyzed the crystal structures of the 35NIDmAb Fab and IFP35 NID complex and the IFP35 NID with a resolution of 2.9 angstroms. In the structure of 35NIDmAb Fab-IFP35 NID IFP35 exists as a monomer. However, in the structure of IFP35 NID alone, IFP35 NID exists as a dimer. In addition, the structures of a single IFP35 NID in these two structures are similar. In the structure of 35NIDmAb Fab-IFP35 NID, there are four 35NIDmAb Fab-IFP35 NID complexes in an asymmetric unit. Each 35NIDmAb Fab is composed of a heavy chain (Ab VH) and a light chain (Ab VL), and interacts with one IFP35 NID. The Fab mainly recognizes and binds to the C-terminal of IFP35 NID. In this interaction interface, the main amino acid residues of IFP35 NID involved are at positions Arg163, Asn164, Arg191, Gln194, Ile195, Gln197, Phe198, Thr199, Pro201, Gln206, Pro208 and Arg210.
[0302] In addition, the present inventors can reveal the key antibody amino acid residues that interact with the antigen based on the structure. The details of the key antibody amino acid residues are as follows.
[0303] In the CDR1 sequence of the heavy chain variable region of the antibody (25 GYTFTNYG 32), the main amino acid residues that bind to an antigen are the two amino acid residues Asn30 and Tyr31. In the CDR2 sequence of the heavy chain variable region of the antibody (50 INTYTGEP 57), the main amino acid residues that bind to an antigen are the three amino acid residues Asn51, Tyr53 and Thr54. In the CDR3 sequence of the heavy chain variable region of the antibody (98 YGYSWAMDY 106), the main amino acid residues that bind to an antigen are the two amino acid residues Tyr100 and Trp102.
[0304] In the CDR1 sequence of the light chain variable region of the antibody (26 SSSVSY 31), the main amino acid residues that bind to an antigen are Ser30 and Tyr31. In the CDR2 sequence of the light chain variable region of the antibody (49 DTS 51), the main amino acid residue that binds to an antigen is Asp49. In the CDR3 sequence of the light chain variable region of the antibody (90 WSSNPPI 96), the main amino acid residues that bind to an antigen are Ser92 and Asn93.
[0305]
The Structure of NMI NID
[0306] In addition to the structures of IFP35 NID and the 35NIDmAb Fab and IFP35 NID complex, the present inventors also analyzed the structure of NMI NID. The overall structure of NMI NID is similar to that of IFP35 NID. The amino acid residues at the corresponding positions of NMI NID are different from those of IFP35 NID compared with the amino acid residues in IFP35 NID involved in the interaction of 35NIDmAb Fab and IFP35 NID (see the sequence alignment diagram below), and the corresponding residues are Arg185, Asn186, Lys215, Lys218, Lys219, Glu221, Tyr222, Pro223, Tyr225, Cys230, Arg232 and Thr234, respectively. These amino acid residues may affect the interaction of NMI NID and 35NIDmab. These NMI amino acid residues represent an antibody binding epitope of NMI, which is helpful for screening and obtaining neutralizing antibodies.
[0307] Alignment of the antibody binding site sequence of IFP35 with the sequence of the homologous segment of NMI:
TABLE-US-00010 IFP35_human156 (SEQIDNO:28) EIFFGKTRNGGGDVDVREL--LPGSVMLGFARDGVAQRLCQIGQFTVPLGGQQVPLRV 211 NMI_human178 (SEQIDNO:29) ELSFSKSRNGGGEVDRVDYDRQSGSAVITFVEIGVADKILKKKEYPLYINQTCHRVTV 235
Experimental Methods
(1) Expression and Purification of IFP35 NID and NMI NID
[0308] The gene fragment encoding IFP35 NID (residues at positions 124 to 220) was cloned into pGEX-6p-1 and expressed in E. coli. The plasmid for encoding IFP35 NID was transformed into BL21 (DE3) cells. 100 mg/L ampicillin was added to LB medium, and the 30 bacteria were cultured at 37 C. When the OD600 reached 0.8 to 1.0, isopropyl--D-thioglactosidase at the final concentration of 0.5 mM was added to induce the expression of IFP35 NID in bacteria at 16 C. for 20h. Then the bacteria were collected by centrifugation at 4000 rpm for 15 minutes. The bacteria were resuspended in lysis buffer (20 Mm Tris at pH 8.0, 400 mM NaCl, 5% Glycerol), subjected to sonication in an ultrasonic apparatus, and then centrifuged at the high speed of 16000 rpm for 30 minutes. The supernatant obtained after centrifugation was added to the GST affinity column for binding for 1 hour, and then the GST affinity column was washed. The recombinant protein was eluted with elution buffer (20 mM Tris at pH 8.0, 150 mM NaCl, 30 mM Glutathione). The eluted protein was digested with PPase, and further purified using an ion exchange column (high salt buffer: 20 mM Tris at pH 8.0, 1M NaCl, 5% Glycerol; low salt buffer: 20 mM Tris at pH 8.0, 100 mM NaCl, 5% Glycerol) and the gel filtration chromatography column Superdex 200 (buffer: 20 mM Tris at pH 8.0, 150 mM NaCl). SDS-PAGE protein gel and Coomassie brilliant blue staining were used to verify the purity and integrity of the recombinant protein.
[0309] The methods of expression and purification of NMI NID (residues at positions 155 to 240) were the same as those of IFP35
(2) Production and Purification of 35NIDmAb Fab
[0310] The 35NIDmAb hybridoma cells and 35NIDmAb antibodies were produced using the methods reported previously. 1 mM EDTA and 1 mM cysteine were added to PBS, and the resultant buffer was used to dissolve papain. The purified 35 NIDmAb was cleaved with papain at a mass ratio of 100:1 (35 NIDmAb: papain) at 37 C. for 6-8 hours to obtain Fab.
(3) Crystallization and Structure Analysis
[0311] During the crystallization process, 35NIDmAb Fab and IFP35 NID were mixed at a ratio of 1:1, and then separated and purified by gel filtration chromatography using superdex 200. The hanging drop method was used for crystal screening. The crystals were dropped at 16 C. with a mixture of 0.2 l protein (10 mg/ml)+0.2 l reservoir solution. The crystals of Fab and IFP35 NID were grown in the reservoir solution containing 0.1 M Tris (pH 8.5), 0.2M MgCl.sub.2, and 18% PEG 3350. 15% DMSO was added to the crystallization solution to protect the crystals at low temperature. The crystals were subjected to crystal diffraction on the beamline BL17U and BL18U of the Shanghai Synchrotron Radiation Facility. HKL3000 was used to process the diffraction data.
[0312] The structures of IFP35 NID and NMI NID were solved by the single-wavelength anomalous dispersion method. SHELX C/D, Phenix.Autosol and Phenix.Autobuild were used to obtain their initial structure models. Coot and phenix were used to correct the structures. The structure of 35NIDmAb Fab-IFP35 NID complex was solved by molecular replacement method using the structures of neutralizing monoclonal antibody YZ23 (PDB code 3CLF) against HIV and IFP35 NID as models. The initial model of the structure was established with Phenix.Autobuild. Coot and phenix were used to correct the structure.
[0313] The study on COVID-19 and influenza virus infection is shown below.
[0314] Materials and Methods
A Method for Detecting IFP35 and/or NMI in the Serum of Human (Patient or Healthy Normal Person)
[0315] (1) Collection of the sample: 4 mL of venous blood was drawn from a fasting subject and placed in a disposable vacuum blood collection tube without anticoagulants, and centrifuged at 3000 r/min for 15 min. After the serum was separated, the serum sample tube was placed in a water bath at 56 C. for at least half an hour to inactivate viruses. Then the sample tube was stored in a refrigerator at 20 C. or 80 C. for further testing, avoiding repeated freezing and thawing.
[0316] (2) Detection of the sample: the concentrations of IFP35 and/or NMI in the serum were detected by the enzyme-linked immunosorbent assay (ELISA). The detection steps were as follows:
[0317] 2.1 Preparation of reagents, a sample and a standard: the sample was slowly dissolved in reagents at a room temperature (18-25 C.). The standard was serially diluted to 5000 pg/mL, 2500 pg/mL, 1250 pg/mL, 625 pg/mL, 312 pg/mL, 156 pg/mL, and 78 pg/mL. The diluent of standard (0 pg/mL) was directly used as a blank control. The control sample for each test should be prepared for immediate use in order to ensure the validity of the test results.
[0318] 2.2 Detection: the wells of standard, the wells of sample to be tested and the wells of blank control were set, respectively. The plate was covered with a film and incubated at 37 C. for 2 hours. The liquid in the wells was discarded and the plate was spun dry. The working solution A to be tested was added to the wells, and the plate was covered with a film and incubated at 37 C. for 1 hour. The liquid in the wells was discarded. The plate was spun dry and washed for 5 times. The working solution B to be tested was added to the wells and incubated at 37 C. for 1 hour. The liquid in the wells was discarded. The plate was spun dry and washed for 5 times. 90 L of the substrate solution was added to each well. The plate was covered with a film, and incubated at 37 C. in the dark for development (the reaction time was 15-25 minutes, when the first 3-4 wells of the standard wells had a clear gradient of blue and the last 3-4 wells did not have a clear gradient of blue, the reaction was stopped). 50 L of stop solution was added to each well to stop the reaction. The order of adding the stop solution should be the same as that of adding the substrate solution. If the color is uneven, the plate was gently shaken to make the solution as homogeneous as possible. The optical density of each well was measured using a microplate reader after ensuring that there are no water droplets at the bottom of the plate and no bubbles in the wells.
[0319] 2.3 Processing of the results: the concentrations of IFP35 and/or NMI in the serum were calculated through the standard curve. SPSS statistical software was used to process the data. The results were expressed as meanstandard deviation. T test was used to analyze the data of the sample and control groups. P<0.05 indicates a difference statistical significance.
Cell Lines and Virus Strains
[0320] HEK293T (ATCC, CRL-11268), A549 (ATCC, CRM-CRL-185) and MDCK (NBL-2) (ATCC CCL-34TM) cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (Gibco). THP1 cells (ATCC, TIB-202TM) and RAW264.7 cells (ATCCTIB-71TM) were cultured in RPMI1640 medium (Gibco, C1875500BT). The 9-day-old chicken embryos free of specific pathogens (SPF) were purchased from Guangdong Wens Dahuanong Biotechnology Co., Ltd. The influenza A virus A/Puerto Rico/8/1934 (H1N1) (PR8) strain was propagated in SPF chicken embryos and purified by sucrose density gradient centrifugation. The virus titer was measured with MOCK cells.
Antibodies and Reagents
[0321] The monoclonal antibody against IFP35 (H00003430-M01) was purchased from Abnova. The antibody against NMI (ab183724) was purchased from abcam. The NS1 antibody against Influenza A virus (sc-130568) was purchased from Santa Cruz. Anti-influenza A virus nucleoprotein antibody (ab128193) was purchased from abcam. E6446 (dihydrochloride) (HY-12756A) (an inhibitor of TLR7/9) and CU-CPT-9b (HY-112051) (an inhibitor of TLR8) were purchased from Med ChemExpress (MCE, USA). An dsRNA inhibitor of TLR3 (614310) was purchased from Millipore. Resatorvid (TAK-242) was purchased from Selleck (USA).
Mouse
[0322] C57BL/6 (B6) wild-type mice were purchased from Guangdong Medical Laboratory Animal Center (GDMLAC). The C57BL/6 mouse model was used to establish NMI.sup./ or IFP35.sup./ homozygous gene knockout mice according to previously published literatures. All mice used in the experiment were 8 to 12 weeks old and gender matched. The mice are kept in an environment free of specific pathogens. The breeding conditions met the standards of the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University.
Plasmid Construction and Transfection
[0323] All vectors were verified by sequencing. Cells were transfected for transient expression in the presence of the ExFect2000 transfection reagent (Vazyme, T202-1). Briefly, 293T cells were transiently transfected with each of plasmids 24 h before virus infection, and then the 5MOI PR8 virus was inoculated into cells in a 6-well plate. After incubated for 1 hour, the cells were washed once with PBS and replaced with normal DMEM medium without FBS. Then the supernatant was collected for the determination of the virus titer, and samples at different time points were collected and analyzed by Western blot electrophoresis.
Real-Time-Fluorescence Quantitative PCR
[0324] A TRIzol LS reagent (USA) was used to extract total RNA from cells or lung tissues. Then the viral genomic RNA (1 g) was reversely transcribed using specific primer U12A: AGCAAAAGAGG (SEQ ID NO: 30). The PrimeScript II first-strand cDNA synthesis kit (Takara) was used to configure the reverse transcription system. Viral mRNA was first purified with an mRNA isolation kit (China Yesen Biotechnology Co., Ltd.), and then was subjected to reverse transcription reaction using oligo (d T) 18 as a primer. The cDNA product, as a template, was amplified by qPCR with SYBR Green Mix (Applied Biosystems). Quantitative PCR reaction was carried out on the Quant Studio5 (Applied Biosystems, Thermo Fisher Scientific). The standard curve method was used to analyze the data.
Enzyme-Linked Immunosorbent Assay
[0325] Murine mIFP35 (E10460m) ELISA kit was purchased from EIAab company. Human IFP35 ELISA kit (OKEH02088) was purchased from AVIVA SYSTEM BIOLOGY. Human hNMI (CSB-EL015 893HU) and murine mNMI (CSB-EL015893M0) ELISA kits were purchased from CUSABIO. The ELISA assays were performed according to the instruction manual. Briefly: 100 l serially diluted standards, test samples, negative and blank controls were respectively added to the corresponding ELISA plates in duplicate. The plates were incubated at 37 C. for 2 hours after covered with the films. The films were removed and the liquids in the wells were discarded. The plates were placed upside down on a paper towel and tapped to discard the remaining liquids. 100 l of biotin-labeled (1) detection antibody was added to each well, and the plates were covered with the films and incubated at 37 C. for 1 hour. The films were removed and the liquids in the wells were discarded. The plates were placed upside down on a paper towel and tapped to discard the remaining liquids. The plates were washed for 3 times with 1wash buffer. The plates were covered with the films and incubated at 37 C. for 1 hour after adding 100 l of HRP-labeled detection secondary antibody to each well. The films were removed and the liquids in the wells were discarded. The plates were placed upside down on a paper towel and tapped to discard the remaining liquids. The plates were washed for 5 times with 1wash buffer. 90 l TMB substrate was added to each well and incubated at 37 C. in the dark for 15-30 minutes. 50 l of stop solution was added to each well, and the OD450 absorbance value was read with an ELISA plate reader.
Histopathology of Lung
[0326] The lung tissues of all the mice were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4 m sections. Then all the sections were stained with hematoxylin and eosin (H&E), and were detected under a microscope for the tissue damage, necrosis, and inflammatory cell infiltration. Different areas randomly selected were photographed.
Serum Sample of Patient
[0327] In the influenza virus study, patient blood and normal volunteer control samples were obtained from Zhongshan School of Medicine in Sun Yat-sen University, the Sixth Affiliated Hospital of Sun Yat-sen University, the First Affiliated Hospital of Sun Yat-sen University, the First Affiliated Hospital of Guangzhou Medical University, Guangdong Maternity and Child Health Hospital and so on. The serums were stored at 80 C. after separation. All the participants signed the informed consent for testing their serum samples and were approved by the human research ethics committee of the relevant hospital (201701093).
Ethical Description
[0328] The animal experiments were carried out in accordance with the Regulations on Administration of Laboratory Animal Affairs approved by the State Council of the People's Republic of China. The animal experiments had been approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University with the license number of SYXK2016-0112. All operations involving influenza viruses were completed in the third-level biosafety laboratory.
Analysis of Data
[0329] Quantitative data were expressed as meanSD, and unpaired Student's t-test was used to determine whether the differences between groups were statistically significant. Log-rank test was used to analyze whether there were significant differences in the survival rates of mice with different treatments. P value less than 0.05 is considered to be statistically different. P value less than 0.001 is considered to be significantly different (#, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001).
Example 6: Identification of IFP35 and/or NMI as Serological Indicators of Novel Coronavirus (COVID-19) Pneumonia
[0330] The present inventors detected the plasma samples of 5 severe/critical diseased patients with novel coronavirus infection collected from designated hospitals for novel coronavirus pneumonia, and have found that the levels of IFP35 and NMI of all patients were significantly increased. Similarly, the same results were obtained by further analysis using more (tens of) patient serum samples. Noteworthily, combined with the clinical manifestations and prognosis of patients, the levels of IFP35 and NMI were highly correlated with the novel coronavirus infection, that is, the higher the levels of IFP35 and NMI, the more severe the patient's clinical manifestations are and the worse the prognosis is (patients with the highest levels of IFP35 and NMI in this study eventually died).
[0331] Other studies of the present inventors had proved (for example, see the following influenza virus research) that inhibiting these inflammatory factors can reduce the degree of inflammatory response, the severity of diseases and death caused by virus infection as the increased amounts of such inflammatory factors in the serum activate the inflammatory response, trigger excessive inflammatory response and so on. It can be inferred from this that the use of drugs (antibody drugs or chemical drugs) to inhibit IFP35 and/or NMI in the blood can inhibit the occurrence of excessive inflammatory response caused by the novel coronavirus COVID-19 infection.
Example 7: Identification of IFP35 and/or NMI as Serological Indicators of Influenza Virus Infection
[0332] Preliminary studies of the present inventors indicated that LPS stimulation induced expression and release of NMI from macrophages. In this study, LPS was used as a positive control, and PBS was used as a negative control.
[0333] For NMI, the amount of NMI in serum of patients infected with influenza A virus is higher than that of healthy donors (Panel A of
[0334] The results observed for IFP35 were similar to those of NMI. The amount of IFP35 in serum of patients infected with influenza A virus is higher than that of healthy donors (Panel B of
[0335]
[0336] All these results indicate that influenza virus infection can lead to the production of NMI/IFP35 in vitro and in vivo.
Example 8: Verification of the Protective Effect of NMI and IFP35 Deletion on Influenza Virus Infection
[0337] NMI.sup./ gene knockout mice were established using the C57BL/6 mouse model. A group of C57BL/6 wild-type or NMI.sup./ gene knockout mice were tested with 210.sup.6 pfu of PR8 virus, which can kill 90% of the infected mice. All mice were monitored daily to determine whether or not they were alive, had weight loss, and had clinical symptoms of the disease (for example, lethargy, hair loss, wrinkled fur, hunched posture, rapid shallow breathing or audible Rales). The daily clinical score of each mouse ranges from 0 (asymptomatic) to 5 (dying). As shown in panels A and B of
[0338] In order to study the homologous protein IFP35, CRISPR Cas9 technology was used to knock out the IFP35 gene in C57BL/6 mice. C57BL/6 wild-type or IFP35.sup./ gene knockout mice were infected with LD.sub.90 dose of PR8 virus respectively for experiments. As shown in
[0339] The above evidences show that reducing the in vivo levels of NMI and/or IFP35 has a protective effect on influenza virus infection.
Example 9: Verification of the Protective Effect of Exogenous Inhibitors on Influenza Virus Infection
[0340] In order to verify whether exogenous inhibition of IFP35 can achieve similar protection, a neutralizing antibody against IFP35 was used as a drug to treat 12 C57BL/6 wild-type mice 1 day before virus infection. At the same time, the same amount of mouse IgG was used to treat another group of C57BL/6 wild-type mice as a negative control. Each mouse was injected intravenously with 200 g antibody for 5 consecutive days, and then all mice were inoculated with LD.sub.90 dose of PR8 virus on day 0. Each mouse was weighed and monitored for wrinkled fur, lethargy, creeping, hunched back, shortness of breath, and audible rales. The clinical symptoms were scored daily for 2 weeks. A schematic diagram of the experimental protocol was shown in Panel A of
[0341] The results showed that: the mice treated with the neutralizing antibody against IFP35 could significantly protect them from the lethal PR8 infection (LD.sub.90 dose), while most of the mice given mIgG died (Panel D of
[0342] Influenza viruses, such as the pandemic H1N1, highly pathogenic H5N1 and novel recombinant H7N9, can directly infect humans and cause severe acute lung injury, acute respiratory distress syndrome, and ultimately respiratory failure. This usually leads to secondary bacterial infections, a variety of infectious pneumonia, encephalitis, myocarditis, and eventually multiple organ failure.
[0343] The influenza virus research of this application supports the following conclusions: influenza virus infection can lead to an increase in the amounts of IFP35 and/or NMI in the blood of infected individuals. This result can be used as a characteristic indicator for detecting influenza virus infection clinically. At the same time, exogenous administration of antibody drugs and chemical drugs for inhibiting IFP35 and/or NMI can be used for the treatment of influenza virus infection.
[0344] Without departing from the spirit and scope of the disclosure of the present application, various modifications and equivalent substitutions can be made to the various embodiments disclosed in the present application. Unless the context indicates otherwise, any features, steps, or embodiments of the present disclosure can be used in combination with any other 20 features, steps, or embodiments.