HUMANIZED MONOCLONAL ANTIBODY FOR 2019 NOVEL CORONAVIRUS AND USE THEREOF
20230073067 · 2023-03-09
Assignee
Inventors
- Jinghua Yan (Beijing, CN)
- Rui SHI (Beijing, CN)
- Qihui WANG (Beijing, CN)
- Fu Gao (Beijing, CN)
- Sufang MA (Beijing, CN)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
G01N2469/10
PHYSICS
G01N33/577
PHYSICS
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
Provided in the present invention are a humanized monoclonal antibody for the 2019 novel coronavirus (2019-nCOV) and the use thereof. The antibody is capable of specifically binding with a receptor binding domain (RBD) of 2019-nCOV and blocking the binding between the RBD of 2019-nCOV and ACE2, and furthermore inhibiting the infection caused by 2019-nCOV.
Claims
1. A human monoclonal antibody or antigen binding fragment thereof that binds to a 2019-nCoV receptor binding domain (RBD), the antibody or antigen binding fragment thereof comprising: a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: (I) heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or (II) HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively, or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; and the light chain variable region comprising: (I) light chain complementarity determining regions (LCDR) LCDR1, LCDR2 and LCDR3 with the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or (II) LCDR1, LCDR2 and LCDR3 with the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences with the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
2. The human monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises: (I) HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or (II) HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; and wherein the light chain variable region comprises: (I) LCDR1, LCDR2 and LCDR3 with the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or (II) LCDR1, LCDR2 and LCDR3 with the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
3. The human monoclonal antibody or antigen binding fragment thereof according to claim 2, wherein HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and wherein LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or wherein HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively, and wherein LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
4. The human monoclonal antibody or antigen binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and wherein the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 31, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto, and wherein the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 32, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto.
5. The human monoclonal antibody or antigen binding fragment thereof according to claim 4, wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7 and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8; or wherein the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 31 and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 32.
6. The human monoclonal antibody or antigen binding fragment thereof according to claim 5, wherein the antibody comprises a heavy chain: comprising an amino acid sequence as shown in SEQ ID NO: 22, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and wherein the antibody comprises a light chain comprising an amino acid sequence as shown in SEQ ID NO: 23, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or wherein the antibody comprises a heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 33, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and wherein the antibody comprises a light chain comprising an amino acid sequence as shown in SEQ ID NO: 34, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
7. The human monoclonal antibody or antigen binding fragment thereof according to claim 6, wherein the heavy chain has an amino acid sequence as shown in SEQ ID NO: 22 and the light chain has an amino acid sequence as shown in SEQ ID NO: 23; or wherein the heavy chain has an amino acid sequence as shown in SEQ ID NO: 33 and the light chain has an amino acid sequence as shown in SEQ ID NO: 34.
8. The human monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, and diabody.
9. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 22 and 23.
10. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 32, 33 and 34, wherein the polypeptide is part of a human monoclonal antibody that binds to a 2019-nCoV receptor binding domain (RBD), and wherein when the polypeptide comprises SEQ ID NO: 31, the human monoclonal antibody further comprises the polypeptide as shown in SEQ ID NO: 32; or wherein when the polypeptide comprises SEQ ID NO: 32, the human monoclonal antibody further comprises the polypeptide as shown in SEQ ID NO: 31; or wherein when the polypeptide comprises SEQ ID NO: 33, the human monoclonal antibody further comprises the polypeptide as shown in SEQ ID NO: 34; or wherein when the polypeptide comprises SEQ ID NO: 34, the human monoclonal antibody further comprises the polypeptide as shown in SEQ ID NO: 33.
11. A polynucleotide encoding the human monoclonal antibody or antigen binding fragment thereof according to claim 1.
12. An expression vector comprising the polynucleotide according to claim 11.
13. A host cell comprising the polynucleotide according to claim 11, wherein the host cell is a eukaryotic cell.
14. A method for preparing the human monoclonal antibody or antigen binding fragment thereof according to claim 1, the method comprising the steps of: expressing the antibody or antigen binding fragment thereof or the polypeptide in the host cell according to claim 13 under conditions suitable for expression of the antibody or antigen binding fragment thereof or the polypeptide, and recovering the expressed antibody or antigen binding fragment thereof or the polypeptide from the host cell.
15. A pharmaceutical composition comprising the human monoclonal antibody or antigen binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier.
16. (canceled)
17. A kit comprising the human monoclonal antibody or antigen binding fragment thereof according to claim 1, or a pharmaceutical composition comprising the human monoclonal antibody or antigen binding fragment thereof and a pharmaceutically acceptable carrier.
18. (canceled)
19. A method for detecting the presence of a 2019-nCoV in a sample, the method comprising the step of contacting the human monoclonal antibody or antigen binding fragment thereof according to claim 1 with the sample.
20. A method for treating and/or preventing a 2019-nCoV infection, the method comprising the step of administering to a subject in need thereof the human monoclonal antibody or antigen binding fragment thereof according to claim 1.
21. The host cell according to claim 13, wherein the eukaryotic cell is a mammalian cell.
22. A method for treating and/or preventing a 2019-nCoV infection, the method comprising the step of administering to a subject in need thereof the pharmaceutical composition according to claim 15.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0083] L. O. D (limit of detection): 200 copies/ml.
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L. O. D (limit of detection): limit of detection, 1000 copies/g.
DETAILED DESCRIPTION OF THE INVENTION
[0086] In order to make the objects, aspects and advantages of the present invention more apparent, a more particular description of the invention will be rendered in detail in the following in combination with specific embodiments and with reference to the drawings.
Example 1: Expression and Purification of 2019-nCoV RBD
[0087] To the 3′ end of the coding region of the 2019-nCoV RBD protein (amino acid sequence as shown in SEQ ID NO: 9) was linked the coding sequence of six histidine tags (hexa-His-tag) and a translation terminator codon, and constructed into pFastBac1 vector (purchased from Invitrogen) by linking EcoRI and XhoI. The linking product was then transformed into DH10Bac competent cells (purchased from Tiangen) for baculovirus recombination. Recombinant baculovirus was extracted, transfected into sf9 cells (purchased from Invitrogen) for packaging of baculovirus, amplified, added to Hi5 cells (purchased from Invitrogen) for expression of the 2019-nCoV RBD protein.
[0088] After purification by nickel ion affinity chromatography (HisTrap TMHP (GE)) and gel filtration chromatography (Superose™ 6 Increase 10/300 GL (GE)), relatively pure target protein can be obtained from the cell culture solution containing the target protein. SDS-PAGE identified a size of 30 KD and the results are shown in
Example 2: Isolation of 2019-nCoV RBD Protein Specific Memory B Cells
[0089] With the informed consent of the discharged person recovering after 2019-nCoV infection, 15 mL of blood was collected and PBMCs were isolated. Isolated PBMCs were incubated at a density of 10.sup.7/mL with 2019-nCoV RBD protein at a final concentration of 400 nM for binding for half an hour on ice, then washed twice with PBS and then incubated with the following antibodies (all purchased from BD): anti-human CD3/PE-Cy5, anti-human CD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC, and anti-His/PE. After half an hour of incubation on ice, PBMCs were washed twice with PBS.
[0090] The PBS-washed PBMCs were sorted by FACSAria III, and cells of PE-Cy5-APC-APC-Cy7+Pacific Blue+FITC+PE+(i.e. B cells) were collected directly into 96-well plates, 1 cell/well.
Example 3: Single B Cell PCR, Sequence Analysis and Human Antibody Design
[0091] According to the method described in Qihui Wang et al. Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus, science Translational Medicine, vol. 8, No. 369, December 2016, the B cells obtained in Example 2 were reverse transcribed by Superscript III reverse transcriptase (Invitrogen) with reverse transcription primers as shown in Table 1 and reacted at 55° C. for 60 min.
TABLE-US-00001 TABLE 1 Reverse transcription reaction primers Primer Sequence No. 5′-3′ sequence IgM-RT SEQ ID NO: 10 ATG GAG TCG GGA AGG AAG TC IgD-RT SEQ ID NO: 11 TCA CGG ACG TTG GGT GGT A IgE-RT SEQ ID NO: 12 TCA CGG AGG TGG CAT TGG A IgA1-RT SEQ ID NO: 13 CAG GCG ATG ACC ACG TTC C IgA2-RT SEQ ID NO: 14 CAT GCG ACG ACC ACG TTC C IgG-RT SEQ ID NO: 15 AGG TGT GCA CGC CGC TGG TC Cκ-new SEQ ID NO: 16 GCA GGC ACA CAA CAG AGG CA RT Cλ-new- SEQ ID NO: 17 AGG CCA CTG TCA CAG CT ext
[0092] 3.1 Human antibody CB6
[0093] Using the above reverse transcription products as templates, antibody variable region sequences were amplified by PCR (PCRa) using HotStar Tap Plus enzyme (QIAgen). The corresponding primers are designed and the reaction conditions are as follows: 95° C. for 5 min; 95° C. for 30 s, 55° C. (heavy chain) for 30 s, 72° C. for 90 s, 35 cycles; 72° C. for 7 min. This product was used as a template for another round of PCR (PCRb) under the following conditions: 95° C. for 5 min; 95° C. for 30 s, 58° C. (heavy chain)/60° C. (κ chain) for 30 s, 72° C. for 90 s, 35 cycles; 72° C. for 7 min, to obtain the PCR products.
[0094] The PCR products were separated by 1.2% agarose gel electrophoresis. Gels with band sizes between 400 and 500 bp were recovered and sent to sequencing. Sequencing results were analyzed using IMGT online software.
[0095] The correct variable region sequence from the analysis were linked with the corresponding heavy/kappa chain constant regions by bridge-PCR and cloned into the expression vector pCAGGS (purchased from Addgene). The heavy chain was linked with EcoRI and XhoI, and the κ chain was linked with SacI and XhoI. B cell sequencing and expression plasmid construction is as follows:
the human antibody design strategy is as follows:
heavy chain: CMV promoter-EcoR I-leader sequence-heavy chain variable region-CH-Xho I; light chain (κ): CMV promoter-Sac I-leader sequence-light chain variable region-CL (κ)-Xho I; wherein, the amino acid sequence of the leader sequence is shown in SEQ ID NO: 18, the amino acid sequence of CH is shown in SEQ ID NO: 19, and the amino acid sequence of CL is shown in SEQ ID NO: 20. By sequence determination, the sequence of an antibody named CB6 was obtained.
[0096] Wherein the heavy chain variable region sequence of CB6 is shown in SEQ ID NO: 7, the light chain variable region sequence is shown in SEQ ID NO: 8, the heavy chain sequence is shown in SEQ ID NO: 22, and the light chain sequence is shown in SEQ ID NO: 23.
[0097] The heavy chain variable region of CB6 has HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region has an amino acid sequence of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Wherein the sequence identity of the CB6 antibody to the germline gene is compared as follows:
TABLE-US-00002 TABLE 2 Comparison of CB6 antibody heavy chain and germline genes V-H allele D-H allele J-H allele Consistency (V-H) CB6 IGHV3-66*01 IGHD3-10*02 IGHJ4*02 99.00%
TABLE-US-00003 TABLE 3 Comparison of CB6 antibody light chain with germline genes V-L allele J-L allele Consistency (V-L) CB6 IGKV1-39*01 IGKJ2*01 99.60%
[0098] 3.2 Human Antibody GH12
[0099] Using the above reverse transcription products as templates, antibody variable region sequences were amplified by PCR (PCRa) using HotStar Tap Plus enzyme (QIAgen). The corresponding primers are designed and the reaction conditions are as follows: 95° C. for 5 min; 95° C. for 30 s, 55° C. (heavy chain)/50° C. (lambda-chain) for 30 s, 72° C. for 90 s, 35 cycles; 72° C. for 7 min. This product was used as a template for another round of PCR (PCRb) under the following conditions: 95° C. for 5 min; 95° C. for 30 s, 58° C. (heavy chain)/64° C. (lambda-chain) for 30 s, 72° C. for 90 s, 35 cycles; 72° C. for 7 min, to obtain the PCR products.
[0100] The PCR products were separated by 1.2% agarose gel electrophoresis. Gels with band sizes between 400 and 500 bp were recovered and sent to sequencing. Sequencing results were analyzed using IMGT online software.
[0101] The correct variable region sequence from the analysis were linked with the corresponding heavy/lambda chain constant regions by bridge-PCR and cloned into the expression vector pCAGGS (purchased from Addgene). Wherein the heavy chain is linked to the lambda chain with EcoRI and XhoI. B cell sequencing and expression plasmid construction is as follows: the human antibody design strategy is as follows:
heavy chain: CMV promoter-EcoR I-leader sequence-heavy chain variable region-CH-Xho I; Light chain (κ): CMV promoter-EcoR I-leader sequence-light chain variable region-CL (κ)-Xho I; wherein, the amino acid sequence of the leader sequence is shown in SEQ ID NO: 18, the amino acid sequence of CH is shown in SEQ ID NO: 19, and the amino acid sequence of CL is shown in SEQ ID NO: 24. By sequence determination, the sequence of an antibody named GH12 was obtained.
[0102] Wherein the heavy chain variable region sequence of GH12 is shown in SEQ ID NO: 31, the light chain variable region sequence is shown in SEQ ID NO: 32, the heavy chain sequence is shown in SEQ ID NO: 33, and the light chain sequence is shown in SEQ ID NO: 34.
[0103] The heavy chain variable region of GH12 has HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively, and the light chain variable region has an amino acid sequence of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
[0104] Wherein the sequence identity of the GH12 antibody to the germline gene is compared as follows:
TABLE-US-00004 TABLE 4 Comparison of GH12 antibody heavy chain and germline genes V-H allele D-H allele J-H allele Consistency (V-H) GH12 IGHV3-7*01 IGHD3-10*01 IGHJ3*02 99.70%
TABLE-US-00005 TABLE 5 Comparison of GH12 antibody light chain with germline genes V-L allele J-L allele Consistency (V-L) GH12 IGLV6-57*02 IGLJ3*02 100.00%
Example 4: Expression of Antibodies
[0105] 293T cells were cultured in DMEM containing 10% FBS. 293T was co-transfected with a plasmid containing genes encoding the light and heavy chains of the specific antibody obtained in Example 3. After 4-6 hours of transfection, the cell culture medium was changed to serum-free DMEM, and the culture was continued for 3 days. After the supernatant was collected, DMEM was added again, and the culture was continued for 4 days, and the supernatant was collected.
[0106] The collected supernatant was centrifuged at 5000 rpm for 30 min, mixed with an equal volume of buffer containing 20 mM sodium phosphate (pH 8.0), filtered through a 0.22 μm filter membrane, and combined with a protein A pre-packed column (5 mL, GE Healthcare). Bound proteins were eluted with 10 mM glycine (pH 3.0). The protein was collected and concentrated and subjected to molecular sieve chromatography. Peaks of interest were determined by SDS-PAGE (reducing and non-reducing) and the results are shown in
Example 5: Surface Plasmon Resonance Detection of Binding Capacity of Antibody to 2019-nCoV RBD
[0107] Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The specific steps are as follows:
protein A chip (purchased from GE Healthcare) was used, the purified antibody obtained in Example 4 was immobilized on the chip by affinity of protein A to Fc of the antibody in an amount of about 5000 RU, and 2019-nCoV RBD protein was diluted with 10 mM HEPES, 150 mM NaCl, pH 7.4 solution in double solution, and loaded one by one from a low concentration to a high concentration. Kinetic profiles of antibody binding to 2019-nCoV RBD are shown in
TABLE-US-00006 TABLE 6 Kinetic constants for antibody binding to 2019-nCoV RBD protein ka (1/Ms) kd (1/s) KD (M) CB6 8.95E+05 7.29E−04 8.15E−10 GH12 2.95E+06 1.73E−02 5.87E−09
Example 6: Detection of Antibody of the Present Invention that Block Binding of 2019-nCoV RBD to ACE2
[0108] The gene encoding hACE2 (amino acid sequence shown in SEQ ID NO: 21) was constructed into pEGFP-N1 vector (purchased from Addgene) by XhoI and BamHI and expressed by fusion with GFP to form pEGFP-hACE2 plasmid. The pEGFP-hACE2 plasmid was transfected into HEK293T cells and GFP expression was observed under fluorescence microscope for 24 h. HEK293T-hACE2 cells were collected in 2×10.sup.5 per reaction and incubated with 2019-nCoV RBD (200 ng/mL) for 30 min at room temperature. After centrifugation at 500×g for 5 min, the supernatant was removed and washed twice with PBS. After incubation with anti-His/APC (anti-His/APC) for 30 min at room temperature and washing twice with PBS, the cell surface fluorescence was detected with BD FACSCanto.
[0109] To examine the blocking effect of CB6 and GH12, the purified antibodies obtained in Example 4 (CB6 and GH12) were incubated with 200 ng/mL of 2019-nCoV RBD at a molar ratio of 10:1 at room temperature for 1 h, respectively, and then incubated with HEK293T-hACE2 cells. The remaining steps are the same as above, using anti-His/APC to detect binding of protein to cells. The antibody blocks binding of 2019-nCoV RBD to HEK293T-hACE2 cells as shown in
Example 7: Detection of Antibody in the Present Invention Neutralizing 2019-nCoV Pseudovirus Infection
[0110] Purified antibodies (CB6 and GH12) obtained in Example 4 were diluted 3-fold starting from 50 μg/mL to a 10.sup.th gradient (2.5 ng/mL), respectively, mixed with 1.6×10.sup.4 TCID.sub.50 VSV-2019-nCoV pseudovirus, incubated for 1 h at 37° C., and then added to 96-well plates pre-inoculated with Huh7 cells (purchased from Basic Medical Cell Center of Peking Union Medical College). After incubation for 4 hours, the culture medium and virus solution were discarded, DMEM medium containing 10% FBS was added, and incubation was continued for 48 hours. The culture solution was discarded, washed once with PBS, added 1× lysis solution (Promega, Luciferase Assay System) to lyse the cells, 10 μL of lysis solution was taken, 50 μL reaction substrate was added, and Promega Luminometers detection was performed. Neutralization ability of the antibody against VSV-2019-nCoV pseudovirus was calculated based on luciferase activity at different concentrations, and the results are shown in
TABLE-US-00007 TABLE 7 Neutralizing effect of antibody on virus from different sources mAbs ND.sub.50 (μg/mL).sup.a CB6 0.00027 GH12 3.27 .sup.aHalf inhibitory concentration
[0111] It can be seen that CB6 and GH12 antibodies can neutralize the 2019-nCoV pseudovirus with high neutralizing activity.
[0112] Taken together, CB6 and GH12 antibodies can serve as human monoclonal antibody with high neutralizing activity against a novel coronavirus (2019-nCoV).
Example 8: Detection of Antibody in the Present Invention Neutralizing 2019-nCoV Live Virus
[0113] This study evaluated the neutralizing effect of CB6 antibody on the 2019-nCoV (SARS-CoV-2) live virus by in vitro neutralization assay.
[0114] 8.1 Reagents
TABLE-US-00008 Name Source COVID-19 SARS-CoV-2: National Institute for Viral Disease BetaCoV/Wuhan/ Control and Prevention IVDC-HB-envF13/2020 Vero E6 cells National Institute for Viral Disease Control and Prevention FBS Gibco DMEM Gibco
[0115] 8.2 Experimental Method
[0116] Vero E6 cells were inoculated at a density of 1×10.sup.5 per well in 96-well culture plates and used after 24 hours at 37° C. In a 96-well tissue culture plate in DMEM medium, 50 μl of a serial 2-fold dilution of CB6 antibody (from 48.8 ng/mL to 100 μg/mL) was added. An equal volume of SARS-CoV-2 working stock containing 200 TCID.sub.50 SARS-CoV-2 was then added for a final viral load of 100 TCID.sub.50. The antibody-virus mixture was incubated for 1 h at 37° C. and then transferred to a 96-well microtiter plate containing octameric fused Vero E6 cells and incubated for 3 days at 37° C. in a CO.sub.2 incubator. SARS-CoV-2 infected cells at 100 TCID.sub.50 or cells cultured in control medium (DMEM+10% FBS) were used as positive control or negative uninfected control, respectively. Cytopathic effect (CPE) was observed and recorded in each well before and after infection. Virus back titration was performed to evaluate the correct virus titration used in the experiment. The 50% neutralizing dose (ND.sub.50) was calculated using Prism software. All experiments were performed in an approved biosafety level 3 facility in accordance with standard operating procedures.
[0117] 8.3 Results and Conclusions
[0118] The neutralizing function of CB6 was assessed by co-culturing cells with SARS-CoV-2 live virus in the presence of different concentrations of CB6. As shown in
[0119] CB6 neutralizes SARS-CoV-2 live virus and reduces the pathological damage of the virus to cells.
Example 9: Evaluation of the Protective Effect of the Antibodies of the Invention on Animals
[0120] 9.1 Experimental Design
[0121] 1) Non-human primate experimental animals are approximately 7-year-old rhesus monkeys (Hubei Tianqin Biotechnology Co. Ltd.), 3 females and 6 males, with body weight of 5.7-10.9 kg and age of 2512-2545 days. Routine pathogen detection and coronavirus viroid detection are completed according to the requirements of experimental animal quality management; the experimental animals meeting the requirements shall be adaptively reared in the laboratory for 3 days;
[0122] 2) The control group, COVID-19 antibody treatment group and prevention group were set up, including three rhesus monkeys (C1, C2 and C3) in the control group, three rhesus monkeys (PA1, PA2 and PA3) in the prevention group and three rhesus monkeys (AC1, AC2 and AC3) in the treatment group. Wherein, the control group received a single injection of PBS 24 h before challenge, while the prevention group received CB6 at a dose of 50 mg/kg 24 h before challenge; the treatment group was injected with CB6 at 24 and 72 h after challenge at a dose of 50 mg/kg.
[0123] 3) The animals were infected by endotracheal intubation, and 1 ml of virus solution was inoculated, and the virus inoculation amount was 1×10.sup.5 TCID.sub.50;
[0124] 4) Parameter collection:
[0125] a. The change of disease course and health score of experimental animals were observed and recorded every day;
[0126] b. After animals were anesthetized at different times, body weight and body temperature were measured, and anal swab, throat swab, alveolar lavage fluid and blood samples were collected;
[0127] c. On day 3 and day 6 post-infection, the chest X-ray images of experimental animals were collected, and the time was synchronized with the observation and sampling;
[0128] d. On day 5, day 6 and day 7 post-infection, one animal was randomly selected for necropsy, and different tissue and organ samples were collected. Histopathology, viral load and immunohistochemical analysis were performed on the samples after inactivation by conventional methods in the laboratory.
[0129] 5) Virus load analysis, blood routine analysis, blood biochemical analysis and neutralizing antibody titer analysis of different samples (nasal swab, throat swab, anal swab, blood, tissue and organ) were completed, imaging of infected animals and pathological analysis of major organ tissues were completed;
[0130] 6) According to the laboratory standard operating procedures and the requirements for biosafety management, all the collected animal tissue and organ samples shall be subjected to histopathological, viral load and immunohistochemical analysis after inactivation treatment in the laboratory; all waste generated during the experiment was disposed according to the waste management regulations of the laboratory.
[0131] 9.2 Experimental Method
[0132] 1) Novel Coronavirus Strains
[0133] Novel coronavirus strain 2019-nCoV-WIV04 (GISAID accession number: EPI_ISI_402124) was isolated by Wuhan Institute of Virology, Chinese Academy of Sciences from a sample of bronchoalveolar lavage fluid from a patient with viral pneumonia in Wuhan in December 2019. After the isolated virus strains are purified, cultured, proliferated and concentrated, 50% tissue cell infective dose (TCID.sub.50) of the virus is determined, and the unit of virus titer is TCID.sub.50/ml.
[0134] 2) Animal Behavior Observation
[0135] During the immunization and virus challenge phase of experimental animals, the skin and hair, secretions, respiration, feces and urine, feed intake and exercise behavior, body weight and temperature were observed daily or at marked time, and the health of each animal was scored according to the “Clinical scoring criteria for rhesus monkeys after novel coronavirus challenge”.
[0136] 3) Virus Titer Determination
[0137] Novel coronavirus samples were inoculated into Vero E6 cells containing DMEM, and after 3 days of culture at 37° C. and 5% CO.sub.2, the culture solution was collected and stored in DMEM for future use. After the isolated virus strains are purified, cultured, proliferated and concentrated, 50% tissue cell infective dose (TCID.sub.50) of the virus is determined. Vero E6 cells were titrated for virus by end-point titration. Ten-fold dilutions of the virus dilutions were inoculated to the cells and after 1 h incubation, the virus dilutions were aspirated and 100 μI DMEM (supplemented with 2% fetal bovine serum, 1 mm L-glutamine, penicillin (100 IU/ml) and streptomycin (100 μg/ml)) was added. Cytopathic score was performed after 3 days of culture and virus titer (TCID.sub.50/ml) was calculated.
[0138] 4) Real-Time Quantitative Fluorescence PCR (qRT-PCR)
[0139] One-step real-time quantitative RT-PCR was used for quantitative analysis of viral RNA in the sample. Viral RNA was extracted from the swabs and blood samples using the QIAamp Viral RNA Mini Kit (Qiagen) according to the supplier's instructions. Samples were homogenized in DMEM (1:10, W/V), centrifuged at low speed at 4500 g for 30 min at 4° C., and RNA was immediately extracted from the supernatant. RNA was eluted in 50 μI of eluate as RT-PCR template. Based on the results of previous studies, primers for the S gene: RBD-qF1: 5′-CAATGGTTAAGGCAGG-3′; RBD-qR1: 5′-CTCAAGGTCTGGATCACG-3′ were used.
[0140] The copy number of RNA in the samples was determined using the HiScript® II One Step qRT-PCR SYBR®. Green Kit (Vazyme Biotech Co., Ltd) kit according to the instruction. 40 cycles of 50° C., 3 min, 95° C., 30 s, including termination at 95° C., 10 s, 60° C., 30 s were performed on an ABI 7700 machine and converted to virus copy number (Copies/ml) according to a standard curve.
[0141] 5) Neutralizing Antibody Assay
[0142] Neutralizing antibody titers in serum samples were determined by neutralizing serum antibodies with a novel coronavirus live virus. The obtained animal serum samples were heat inactivated at 56° C. for 30 min, diluted to 1:50, 1:150, 1:450, 1:1350, 1:4050 and 1:12150, added with equal amount of live virus, and incubated in an incubator containing 5% CO.sub.2 at 37° C. After 1 h incubation, 100 microliters of the mixture were inoculated onto a monolayer of Vero cells in a 12-well plate and incubated for 1 h with shaking every 15 min. After removing the remaining inoculum, a culture medium of DMEM containing 0.9% methylcellulose and 2% FBS was added and incubated at 37° C., 5% CO.sub.2 for 3 days. After 3 days, the cells were fixed with 4% formaldehyde for 30 min, the fixing solution was removed, rinsed with tap water, and then stained with crystal violet. The number of plaques was counted and the neutralizing antibody titer (EC.sub.50) was determined.
[0143] 6) Laboratory Animal Procedures
[0144] All challenge experiments were performed in a Level IV biosafety laboratory. Rhesus monkeys were anesthetized and inoculated with 1 ml of virus solution by endotracheal intubation Animals will be observed and recorded daily for clinical symptoms including the nature, incidence, severity and duration of any severe or visible changes. Pulmonary X-ray was performed with HF100Ha (MIKASA, Japan) at different times to obtain pulmonary images and swab samples and blood samples from oropharynx, turbinate and anal region. The collected swab samples were placed in 1 ml of Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 IU/ml) and streptomycin (100 μg/ml). Whole blood was stored in K2 EDTA tubes for viral RNA extraction and blood routine analysis. To study the pathogenesis and pathological damage of respiratory tract, one animal was randomly euthanized on day 5, 6 and 7 after challenge. The trachea, right bronchus, left bronchus, six lung lobes and other tissues and organs were collected for pathological, virological and immunological analysis.
[0145] 7) Animal Anatomy and Pathological Analysis
[0146] Animal anatomy was performed according to the standard procedures for experimental animals The collected organs and tissue samples were fixed in 10% neutral formalin buffer and then removed from the laboratory for paraffin embedding and sectioning. The sections were observed under light microscope after staining with hematoxylin and eosin. To detect the distribution of the novel coronavirus, paraffin-dehydrated tissue sections were placed in antigen buffer, blocked with 5% bovine serum albumin for 1 h at room temperature, and then blocked with a self-made primary antibody (rabbit anti-RP3-RP3-CoV N protein polyclonal antibody) at 1:500. After washing with PBS, sections were dried slightly, diluted at 1:200, and overlaid with Cy3-conjugated goat anti-rabbit IgG (Abcam) secondary antibody. Slides were washed with PBS and stained with 1:100 diluted DAPI Images were acquired by the Pannoramic MIDI system (3DHISTECH, Budapest, Hungary).
[0147] According to the pathological changes in the lungs of experimental animals, determining the damage level of organs and tissues, wherein: “+++” indicates severe lesion; “++” is moderate lesion; “+” is mild lesion; “−” is no lesion and “+/−” is between mild and no lesion.
[0148] 8) Other Operations
[0149] According to the requirements for management system documents of biosafety level IV laboratory, use the standard operating procedures of laboratory to complete.
[0150] 9.3 Experimental Results
[0151] 1) Selection and Adaptive Feeding of Experimental Animals
[0152] The experimental animals met the requirements for quality management of experimental animal in Hubei Province. The behavior, health and eating of the experimental animals were not abnormal after completing the three-day adaptive feeding in the laboratory.
[0153] 2) Observation on Clinical Symptoms of Experimental Animals after Virus Inoculation
[0154] The animals in control group, treatment group and prevention group were transferred to P4 laboratory. After 3-day adaptive feeding in the laboratory, the following operations were performed respectively.
[0155] Control and prevention groups: PBS and monoclonal antibody (up to 50 mg/kg) were intravenously injected indoors according to the plan. After 24 h, the animals were challenged by endotracheal intubation. Routine observation, body temperature and weight test were performed according to the experimental plan.
[0156] Treatment group: after 24 h challenge by endotracheal intubation, the drug was administered at day 1 and day 3 as planned, and routine observation, body temperature and body weight test were performed according to the experimental plan.
[0157] Results showed that the experimental animals of the control group, the COVID-19 antibody-treated group and the prevention group did not show significant behavioral differences throughout the experimental period, without significant changes in body temperature (see
[0158] 3) Changes in Viral Load of Throat Swabs, Nasal Swabs and Anal Swabs
[0159] Throat swab, nasal swab and anal swab samples were collected daily to detect the change of viral load in different samples. Results showed that the virus load of throat swab in control group changed with the time of infection, and the virus load of experimental animals (C1, C2 and C3) reached the peak on the day 2 to day 4 after inoculation, and then decreased continuously. By day 7, the viral load in the samples dropped to a lower level. However, the virus load of throat swabs varied among different animals, and the peak of virus replication in C1, C2 and C3 was on the day 2, 3 and 4 after challenge, respectively. Overall, however, the variation of the viral load in the throat swab samples showed a proliferation process of the virus in vivo (see
[0160] There were significant individual differences in COVID-19 antibody treatment group; however, all reached the peak value of viral load at day 2 after challenge, and then continued to decrease, the viral load was far lower than that in the control group; the viral load of AC1 individuals at day 3 is lower than the lower limit of detection; Nasal swab samples, all groups had lower viral load; Low levels of viral nucleic acid copy number early in challenge were only detected in nasal swab samples and by day 7 no viral nucleic acid was detected in all nasal swab samples (see
[0161] 4) Imaging Analysis of Infected Animals
[0162] X-ray lung images of 3 animals in the control group on day 0 of challenge were clear, and no pulmonary shadow was observed. On the day of virus challenge, no obvious shadow was observed in the lungs of experimental animals in the control group; On day 3 of challenge, one animal (C3) had a slightly blurred/disturbed lung texture, the other two animals (C1 and C2) had significant pulmonary shadow, and by day 6, no significant pulmonary shadow was seen (see
[0163] 5) Pathological Change of Infected Animals
[0164] On day 5, 6 and 7 after infection, one animal in each of control group, prevention group and treatment group was randomly selected for dissection, and organs and tissues such as lung, trachea, bronchus, heart, liver, spleen, kidney, gastrointestinal tract, reproductive organs and lymph nodes were collected. According to the experimental requirements, the shape, color and tissue lesion site of each organ were observed.
[0165] On day 5 after challenge, animals in control group (C2) had light and off-white color on the general lung surface, moderate and slightly severe bilateral lower lung lesions, emphysema at the edge of lung lobe, and no obvious abnormality in trachea and bronchus. On day 6 and day 7 after challenge, animals in control group (D6: C3; D7: C1) had dark red general lung surface, moderate and slightly severe bilateral lower lung lesions, emphysema at the edge of lung lobe was mixed with lung consolidation area, and no obvious abnormality in trachea and bronchus.
[0166] On day 5 after challenge, animals in the prevention group (PA2) and treatment group (AC2) had dark red general lung surface, moderate and slightly severe bilateral lower lung lesions, obvious lung lobe edge emphysema, and no obvious abnormality in trachea and bronchus. On day 6 and day 7 after challenge, experimental animals (prevention group: D6: PA3, D7: PA1; treatment group: D6: AC3, D7: AC1) had light and off-white color on the general lung surface, moderate and no obvious abnormality in lung, trachea and bronchus.
[0167] The histopathological changes of the lungs showed that on day 5 after infection, the changes of pulmonary diffuse interstitial pneumonia, alveolar wall thickening, fibroblast proliferation, fibrosis, and infiltration of monocytes and lymphocytes were observed in the control group; some alveolar edema and fibrin exudation, transparent membrane formation and pulmonary hemorrhage were observed. Transparent thrombosis was formed in part of pulmonary capillary lumen, and epithelial cells of bronchioles were necrotic and exfoliated. On day 6 and 7 after infection, pulmonary edema and fibrin exudation increased, alveolar macrophages increased, alveolar wall and alveolar fibrosis and organization were obvious. Compared with the control group, no matter the treatment group or the prevention group, the experimental animals had less lung injury caused by novel coronavirus. On day 5 after infection, the alveolar structures of two animals (AC2 and PA2) were basically intact, focal or patchy pulmonary fibrosis was observed, and mononuclear cells and lymphocyte infiltration were observed, but the number was significantly reduced compared with the control group, there were more macrophages in alveolar cavity, less edema, no transparent membrane formation, and no severe small bronchioles and pulmonary capillary lesions. On day 6 and 7 after infection, the pathological changes of animal lungs were significantly improved, the exudative lesions of lung tissue basically disappeared, and focal or patchy tissue fibrosis could be seen.
[0168] 6) Viral Load in Different Organ Tissues
[0169] In order to further understand the distribution of virus in different tissues of upper respiratory tract and lung, the tissues of different parts of trachea, bronchi and lung of control group and two antibody group animals were collected on day 5, 6 and 7 after challenge, respectively, and the viral load in different organs and tissues was determined. The results showed that on day 5 after challenge, 4-8×10.sup.5 viral copies/g were detected in trachea and left bronchi of only one animal in prevention group (AC2), while no virosome was detected in other tissue samples, and no viral nucleic acid was detected in all treatment group animal samples (see
[0170] As shown in
[0171] 7) Immunohistochemical Analysis
[0172] The results of immunohistochemical analysis of the virus in the lung tissue showed that the virus protein could be detected in the lungs from the control group on day 5, 6 and 7. However, the virus protein could not be detected in the lungs from the antibody treatment group and prevention group at different time.
[0173] The specific embodiments described above further illustrate the objects, technical solutions and advantages of the present invention. It should be understood that the foregoing description is only illustrative of specific embodiments of the invention, and is not intended to limit the invention. It is intended that the invention be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.