CN117467668B

CN117467668B - Neutralizing antibody for resisting severe acute respiratory syndrome type II coronavirus SARS-COV-2

Info

Publication number: CN117467668B
Application number: CN202211595499.6A
Authority: CN
Inventors: 刘密; 王义鹏; 刁璐; 马琳; 镇学初; 龙亚秋
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-11-25
Filing date: 2021-10-20
Publication date: 2024-06-21
Anticipated expiration: 2041-10-20
Also published as: CN116396967A; CN117467668A; CN113801223A; CN112442120A; CN116254270A; CN116003584A; WO2022110962A1; CN116396967B; CN113801223B

Abstract

The invention discloses a neutralizing antibody for resisting severe acute respiratory syndrome type II coronavirus SARS-COV-2, belonging to the technical field of biological medicine. The invention extracts peripheral immune cells from COVID-19 blood of a rehabilitation patient, screens B cells which can be combined with a novel coronavirus antigen protein-spike protein, and analyzes single B cells for producing antibodies at a single cell level to obtain the gene sequences of the heavy chain and the light chain of the neutralizing antibody variable region in the B cells. The sequences can be used for reconstructing and expressing neutralizing antibodies capable of neutralizing the new coronavirus in vitro, and are expected to be used for treating and preventing diseases such as pneumonia and the like caused by the new coronavirus.

Description

Neutralizing antibody for resisting severe acute respiratory syndrome type II coronavirus SARS-COV-2

The application is as follows: 202111222859.3, the application date is: 2021, 10-20, application name: a divisional application of neutralizing antibodies against severe acute respiratory syndrome type II coronavirus SARS-COV-2.

Technical Field

The invention relates to a neutralizing antibody for resisting severe acute respiratory syndrome type II coronavirus SARS-COV-2, belonging to the technical field of biological medicine.

Background

Pneumonia (COVID-19) caused by infection with severe acute respiratory syndrome type II coronavirus (SARS-CoV 2) is a serious infectious disease and causes serious effects on a global scale. Finding an effective therapeutic regimen for this virus is an urgent need.

The neutralizing antibody is an antibody which can eliminate the virus infection ability after being combined with the virus, is a corresponding antibody generated by B lymphocyte when pathogenic microorganism invades the organism, and is a soluble protein secreted by adaptive immune response cell. Pathogenic microorganisms invade cells and require specific molecules expressed by the pathogen itself to bind to receptors on the cells to infect the cells and further expand. The neutralizing antibodies are capable of binding to antigens on the surface of the pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to the target cell receptor and from invading the cell. After the virus invades the human body, the B cells secrete neutralizing antibodies into the blood, and the antibodies bind to the virus particles in the blood to prevent the virus from infecting the cells and damaging the virus particles, so that the virus is "neutralized". It follows that neutralizing antibodies play a major role in killing free viruses outside of cells.

Certain specific neutralizing antibodies from the blood of a patient cured from a viral infection have the effect of neutralizing the virus and are therefore useful in the treatment of infectious diseases, rendering the virus pathogenic. With advances in antibody production technology, therapeutic antibodies have gradually performed well in the treatment of a variety of diseases. Existing vaccines such as measles vaccine, poliomyelitis vaccine, hepatitis B vaccine and hepatitis A vaccine all enable an inoculator to generate neutralizing antibodies to prevent virus infection. Since neutralizing antibodies destroy viruses before they enter cells and remove free viruses from the cells in vivo, neutralizing antibodies in the blood of patients recovering from viral infection can be used in the treatment of viral infection.

Disclosure of Invention

In order to solve the technical problems, the invention extracts peripheral immune cells from the blood of COVID-19 rehabilitation patients, screens B cells which can be combined with new coronavirus antigen protein-spike protein, and analyzes single B cells for producing antibodies at a single cell level to obtain the gene sequences of the heavy chain and the light chain of the neutralizing antibody variable region in the B cells. The sequences can be used for reconstructing and expressing neutralizing antibodies capable of neutralizing the new coronavirus in vitro, and are expected to be used for treating and preventing diseases such as pneumonia and the like caused by the new coronavirus.

It is a first object of the present invention to provide a neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2 comprising: a light chain variable region DNA sequence and a heavy chain variable region DNA sequence;

The nucleotide sequence of the light chain variable region DNA sequence is one or a combination of a plurality of SEQ ID NO. 1-5;

The nucleotide sequence of the heavy chain variable region DNA sequence is one or more of SEQ ID NO. 6-8.

Further, the neutralizing antibody is a whole antibody comprising a constant region and a variable region, a partial antibody comprising only a variable region, or a chimeric antibody comprising only a variable region.

It is a second object of the present invention to provide a gene encoding the neutralizing antibody.

A third object of the present invention is to provide an expression vector carrying the gene.

It is a fourth object of the present invention to provide a recombinant cell expressing said neutralizing antibody.

It is a fifth object of the present invention to provide the use of said neutralizing antibodies for the preparation of a medicament for the treatment of COVID-19.

A sixth object of the present invention is to provide a kit for treating pneumonia COVID-19, wherein said kit contains said neutralizing antibody.

The beneficial effects of the invention are as follows:

The invention extracts peripheral immune cells from COVID-19 blood of a rehabilitation patient, screens B cells which can be combined with a novel coronavirus antigen protein-spike protein, and analyzes single B cells for producing antibodies at a single cell level to obtain the gene sequences of the heavy chain and the light chain of the neutralizing antibody variable region in the B cells. The sequences can be used for reconstructing and expressing neutralizing antibodies capable of neutralizing the new coronavirus in vitro, and are expected to be used for treating and preventing diseases such as pneumonia and the like caused by the new coronavirus.

Drawings

FIG. 1 is a schematic of a flow chart for screening neutralizing antibodies using COVID-19 to rehabilitate single B cells in a patient's blood;

FIG. 2 shows the results of an antibody experiment after expression purification by SDS-PAGE analysis;

FIG. 3 shows the results of a pseudo-virus neutralization assay for negative control antibody Pmab (no neutralizing effect) and neutralizing antibody HC5K VH/B5K VL (IC ₅₀ -0.1273. Mu.g/mL);

FIG. 4 shows the result of neutralization experiments (IC ₅₀ -0.00033. Mu.g/mL) of neutralizing antibody D5K VH/B5K VL pseudovirus;

FIG. 5 shows the results of a neutralization experiment of neutralizing antibody HC5K VH/D4K VL (IC ₅₀ -0.00084. Mu.g/mL) and neutralizing antibody HF4L VH/D5K VL (IC ₅₀ -0.0046. Mu.g/mL) pseudoviruses;

FIG. 6 shows the results of neutralization experiments of neutralizing antibody HC5K VH/D5K VL (IC ₅₀ -0.0066. Mu.g/mL) and neutralizing antibody HD5K VH/A5K VL (IC ₅₀ -0.0017. Mu.g/mL) pseudoviruses;

FIG. 7 shows the results of a neutralization experiment of neutralizing antibody HF4L VH/A5K VL (IC ₅₀ -0.6049. Mu.g/mL) and neutralizing antibody HF4L VH/B5K VL (IC ₅₀ -0.6077. Mu.g/mL) pseudoviruses;

FIG. 8 shows the result of neutralization experiment of pseudovirus of neutralizing antibody HD5K VH/D4K VL (IC ₅₀ -0.1996. Mu.g/mL).

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Example 1: isolation and extraction of single B cell capable of combining with SARS-COV-2

And (3) extracting about 15mL of blood of a plurality of new coronavirus rehabilitation patients, separating and extracting Peripheral Blood Mononuclear Cells (PBMC) in the blood of each rehabilitation patient by adopting a Ficoll gradient method, and washing the obtained PBMC twice for later use.

Fc block was added to each PBMC sample, and after 15 minutes of action, APC-H7 labeled anti-human CD3 antibody, BV421 labeled anti-human CD19 antibody, BB700 labeled anti-human CD27 antibody, biotin labeled SARS-COV-2Spike protein, and Biotin labeled SARS-COV-2Spike RBD were added sequentially. Strepitavidin-APC was then added and allowed to act for 30 minutes. And then, carrying out loading analysis by adopting a FACS ARIATMIII flow cytometer to collect the PBMC sample marked by the antibody. CD3 is a specific surface marker of T cells, CD19 is a specific surface marker of B cells, CD27 is a specific surface marker of memory B cells, and two antigens marked by Biotin can be specifically combined with strepitavidins marked by APC, so that the APC positive cell is the cell capable of being specifically combined with SARS-COV-2. The cell selected from CD3 ^-CD19⁺CD27⁺APC⁺ is the memory B cell which can be specifically combined with SARS-COV-2. The B cells are separated by a flow cytometer, directly injected into a 96-well plate preloaded with cell lysate, immediately sealed, rapidly frozen on dry ice, and then preserved at-80 ℃ for later use. Thus, single B cells which can specifically bind to SARS-COV-2 can be isolated.

Example 2: amplification and sequencing of variable region light and heavy chains in single B cells

Since the B cells are antibody-secreting cells, the sequence information of the antibodies is stored in the B cells that bind to SARS-COV-2. Single B cell lysates that had been lysed in 96-well plates were divided into 3 parts. One was used to perform Kappa light chain sequence analysis, one was used to perform lamda light chain sequence analysis, and the other was used to perform heavy chain sequence analysis.

In this part of the experiment, a cDNA library was obtained by reverse transcription of RT-PCR, and then the DNA sequence in a single cell was amplified by using nested PCR technique. Because the primers used in the experiments were primers specific for either the light chain variable region or the heavy chain variable region, the resulting sequences were the sequences of the light chain and heavy chain portions of the antibody variable region in the single B cell. And (3) carrying out DNA sequencing analysis on the amplified sequences to obtain the sequence information of the light chain and the heavy chain of the antibody variable region contained in the single B cell.

RT-PCR amplified single B cell antibody light-heavy chain variable region gene

RT-PCR primers (SEQ ID NOS.9-25) were designed as shown in Table 1:

TABLE 1

Single B cell RT-PCR was performed using the single B cells selected as templates. The preparation and sample addition of the PCR system are completed in a biosafety cabinet and are put on ice for operation, and the RT-PCR reaction system and reaction conditions are as follows:

PCR reaction system:

TABLE 2

Composition of the components	System of	Final concentration
			RNase-free water	-	-
5 XRT-PCR buffer	10.0μl	1×
			DNTP mixture	2.0μl	400. Mu.M of each dNTP
A primer	-	0.6μM
			B primer	-	0.6μM
Mixed enzyme	2.0μl
			RNase inhibitors	-	5-10 Units
Template RNA	-	1pg–2μg
			Total system	50.0μl	-

PCR reaction conditions:

nest PCR amplified single B cell antibody light-heavy chain variable region gene

Nested PCR primers (SEQ ID NOS.26-38) were designed as shown in Table 3:

TABLE 3 Table 3

Three PCR reaction systems are established by taking RT-PCR products as templates, variable region genes of H chains, kappa chains and lambda chains of antibodies are amplified respectively, and each reaction system uses mixed primers corresponding to the reaction systems respectively, wherein the nested PCR reaction systems and reaction conditions are as follows:

PCR reaction system:

TABLE 4 Table 4

PCR reagent	Volume (μl) of each sample (25 μl system)	Final concentration
			Taq DNA polymerase	0.25	50U ml^-1
10 Xbuffer	2.5	1×
			dNTPs(10 mM)	0.5	200μM
Forward primers VH3a and VH3b or PanV κ	0.5	1.2μM
			Reverse primer PW-Cgamma or CK494-516	0.5	1.2μM
RNase-free water	17.25-19.25 (Filling in System to 24. Mu.l)	-
			Template	1.0	-

PCR reaction conditions:

example 3: experimental results

The application obtains 5 light chain variable region sequences and 3 heavy chain variable region sequences from B cells of recovered patients capable of combining new coronavirus spike protein. The 5 light chain variable region sequences and 3 heavy chain variable region sequences can be freely combined into an antibody, namely the neutralizing antibody of the novel coronavirus SARS-COV-2.

Among them, 5 light chain variable regions were named A5K VL (SEQ ID NO. 1), B5K VL (SEQ ID NO. 2), D4K VL (SEQ ID NO. 3), D5K VL (SEQ ID NO. 4) and D7K VL (SEQ ID NO. 5), respectively, and 3 heavy chain variable regions were named HD5K VH (SEQ ID NO. 6), HF4L VH (SEQ ID NO. 7) and HC5K VH (SEQ ID NO. 8), respectively.

The expression and purification of the antibodies were performed as follows:

This example constructed 9 antibodies, ：HC5K VH/B5K VL、HD5K VH/D4K VL、HF4L VH/A5K VL、HF4L VH/B5K VL、HD5K VH/B5K VL、HC5K VH/D4K VL、HF4L VH/D5K VL、HC5K VH/D5K VL and HD5K VH/A5K VL, respectively.

1. Vector construction

Primers were designed according to the corresponding antibody light and heavy chains, and the corresponding VH and VL sequences were amplified. The amplified VH and VL sequences were recombined onto antibody expression vector backbone plasmids using a recombinase (ClonExpress II One Step Cloning Kit, C112-01/02, vazyme), and bacterial fluids were sequenced.

2. Plasmid extraction

The correct clone was inoculated into LB (Amp 100 ug/ml) medium, cultured overnight at 37℃and the plasmid was extracted with a plasmid pump kit the next day and the concentration was determined.

3. Transient cell expression

The extracted plasmid is transiently transformed into 293F cells, and the method is as follows:

1) 293F in the logarithmic growth phase was counted and cells were resuspended in fresh 293 medium to a density of 2X 10 ⁶/mL and a total volume of 100mL.

2) 100Ug of plasmid was diluted with medium.

3) PEI was diluted with medium (1. Mu.g/. Mu.L) and the diluted PEI was added to the diluted plasmid DNA. Mixing is performed by rotating and/or inverting the tube or gently swiping 2 to 3 times with a pipette. The complex was incubated at room temperature for about 20 minutes.

4) The plasmid/PEI mixture was added to the cell suspension and the flask was gently shaken during the addition and incubated at 37℃in 8% CO ₂ at 125 rpm.

5) 5% (V/v) of the feed was added to the shake flask 16-22 hours after transfection, and the shake flask was gently shaken during the addition and returned to the 37℃incubator.

6) Supernatants were harvested on day 6.

4. Antibody purification

1) Harvesting the supernatant

The supernatant was harvested by centrifugation using a centrifuge. The supernatant was filtered through a 0.4um filter membrane and the purification column was interfaced.

2) Protein purification

Selecting Protein A affinity filler Mabselect SuReTM of GE company, removing the components of the culture medium, and enriching the target Protein.

5. Purification of the resulting proteins by SDS-PAGE analysis

The resulting antibodies were purified by SDS-PAGE electrophoresis. The concentration of the separation gel used during the analysis was 4-20%. The sample is firstly reduced, then the reduced sample is added into Loading Buffer and then boiled at 70 ℃ for 10min, and then the sample is loaded and analyzed. The results are shown in FIG. 2.

Example 4: pseudovirus neutralization assay

The experiment carried out the evaluation of the new coronaneutralizing antibodies with pseudoviruses carrying the new coronavirus spike protein. The virus system takes HIV-1 carrying a luciferase reporter gene as a virus skeleton, simultaneously expresses new coronavirus Spike protein on a virus shell, and forms pseudovirions to infect exogenous cell lines with high expression of ACE2, so that the invasion process of the new coronavirus to target cells through Spike-ACE2 is highly simulated, and the degree of infection of the target cells by the pseudovirions is positively correlated with the luminous value of luciferase and negatively correlated with the neutralizing activity of antibodies.

The neutralizing antibody is combined with S protein of a virus shell in vitro, so that the site of the S protein combined with ACE2 is blocked, the S protein cannot be combined with the ACE2, and the virus cannot invade cells; on the contrary, antibodies without neutralizing activity cannot interfere the binding of the S protein to the ACE2 on the cell surface, viruses entering the cells express the Fluc protein, and after reacting with a luminescent substrate, the luminescent value is detected by an enzyme-labeled instrument.

The experimental steps are as follows:

1. Sample preparation: pseudoviruses were thawed in advance from-80 ℃ to 4 ℃ in a refrigerator or on ice, and the viruses were diluted to 1-2 x 10 ⁴TCID₅₀/mL with DMEM medium containing 10% FBS prior to use (best TCID50 required to be self-futile if infected ACE2 overexpressing cell lines were purchased from non-me).

2. A new 96-well cell culture plate was taken for sample dilution.

3. 135. Mu.L of the sample to be tested was added to column 2 of the 96-well cell culture plate.

4. The 3 rd to 10 th columns were each filled with 90. Mu.l of serum-free DMEM, then 45. Mu.l of the dilution was added from the 2 nd column to the 3 rd column for double dilution with a row gun until the dilution was 9 th column, and the last column was discarded with an excess of 45. Mu.l. Then 90. Mu.l of diluted pseudovirus solution was added to each well. The Virus Control (VC) was added with 90. Mu.L of pseudovirus and the Cell Control (CC) was added with 180. Mu.L of serum-containing DMEM medium alone, and the 96-well cell culture plates were incubated in a 37℃incubator for 1 hour.

5. Sample-pseudovirus mixtures in 96-well cell culture plates and two control groups were aliquoted into three-well 96-well white plates, 50 μl/well (3 replicates).

6. Immediately, 50. Mu.l of 293-ACE2 cells at a density of 0.4X10 ⁶ cells/ml (number of 2X 10 ⁴ cells/well) were plated in 96 Kong Baiban (without medium change) and incubated in a 37℃incubator.

7. After incubation for 20-24h, 25. Mu.L of DMEM medium containing 10% FBS, pre-warmed at 37℃was added to each well.

8. After further culturing for 48h, 96 Kong Baiban parts are taken out and balanced to room temperature, 125 mu l of Bio-Lite reporter gene detection reagent after balancing at room temperature is added into each hole, the plates are shaken for 2min, and after standing for 5min at room temperature, a chemiluminescent value (RLU) is detected by an enzyme-labeled instrument.

9. And (3) data processing: log values of antibody concentration and corresponding RLU were taken into graph pad software, and IC50 values were calculated and compared.

The experimental results are shown in table 5 and fig. 3 to 8: the neutralizing antibody provided by the invention has a certain neutralizing capacity on the novel coronavirus. However, the neutralizing ability of each antibody against the novel coronavirus varies. The lower the IC50 value in the virus neutralization experiment, the better, indicating that fewer antibodies are needed to neutralize the virus. The IC50 values of the neutralizing antibodies of the novel coronaviruses have been reported to be in the range of 0.5 μg/mL to 0.0012 μg/mL in virus neutralization experiments. In the novel coronavirus neutralizing antibodies of the invention, the IC50 of some antibodies such as HD5K VH/B5K VL and HC5K VH/D4K VL in virus neutralization experiments reaches 0.00033 mug/mL and 0.00084 mug/mL, and the neutralizing capacity of the antibodies to viruses is stronger than that of most novel coronavirus neutralizing antibodies.

TABLE 5 neutralizing antibody Virus neutralization Capacity

Antibodies to	Virus neutralization test IC ₅₀ (μg/mL)
		HC5K VH/B5K VL	0.1273
HD5K VH/D4K VL	0.1996
		HF4L VH/A5K VL	0.6049
HF4L VH/B5K VL	0.6077
		HD5K VH/B5K VL	0.00033
HC5K VH/D4K VL	0.00084
		HF4L VH/D5K VL	0.0046
HC5K VH/D5K VL	0.0066
		HD5K VH/A5K VL	0.0017

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions or transformations, such as within 3% of a base substitution of an antibody variable region heavy or light chain sequence, or within 3% of an amino acid substitution of an amino acid sequence of an antibody portion expressed by an antibody variable region heavy or light chain sequence, are within the scope of the present invention, as would be recognized by one of skill in the art based on the present invention. The protection scope of the invention is subject to the claims.

Claims

1. A neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2 comprising: a light chain variable region DNA sequence and a heavy chain variable region DNA sequence;

the nucleotide sequence of the light chain variable region DNA sequence is SEQ ID NO.3;

The nucleotide sequence of the heavy chain variable region DNA sequence is SEQ ID NO.8.

2. The neutralizing antibody of claim 1 wherein said neutralizing antibody is a whole antibody comprising constant and variable regions, a partial antibody comprising only variable regions, or a chimeric antibody comprising only variable regions.

3. A gene encoding the neutralizing antibody of claim 1 or 2.

4. An expression vector carrying the gene of claim 3.

5. A recombinant cell expressing the neutralizing antibody of claim 1 or 2.

6. Use of a neutralizing antibody according to claim 1 or 2 in the manufacture of a medicament for the treatment of pneumonia COVID-19.

7. A kit for treating pneumonia COVID-19, comprising a neutralizing antibody according to claim 1 or 2.