CN113801223B

CN113801223B - Neutralizing antibody against severe acute respiratory syndrome type II coronavirus SARS-COV-2

Info

Publication number: CN113801223B
Application number: CN202111222859.3A
Authority: CN
Inventors: 刘密; 王义鹏; 刁璐; 章铭辉; 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: 2023-02-10
Anticipated expiration: 2041-10-20
Also published as: CN116396967B; CN112442120A; CN116254270A; CN117467668B; CN117467668A; CN116396967A; WO2022110962A1; CN116003584A; CN113801223A

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

Description

Neutralizing antibody 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 of severe acute respiratory syndrome type II coronavirus (SARS-CoV 2) is a serious infectious disease and causes serious influence on the global scale. It is an urgent need to find an effective treatment for this virus.

The neutralizing antibody refers to an antibody capable of eliminating the ability of viral infection after binding to a virus, is a corresponding antibody produced by B lymphocytes when pathogenic microorganisms invade the body, and is a soluble protein secreted by adaptive immune response cells. Invasion of cells by pathogenic microorganisms requires the binding of specific molecules expressed by the pathogen itself to receptors on the cells in order to infect and further amplify the cells. The neutralizing antibody can bind to an antigen on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to a target cell receptor and invading cells. After the virus invades the human body, B cells secrete neutralizing antibodies into the blood, and the antibodies are combined with virus particles in the blood to prevent the virus from infecting the cells and destroying the virus particles, so that the virus is neutralized. It follows that neutralizing antibodies play a major role in killing extracellular free virus.

Certain specific neutralizing antibodies derived from the blood of a patient cured of a viral infection have a virus-neutralizing effect and are therefore useful in the treatment of infectious diseases, rendering the virus non-pathogenic. With the advances in antibody production technology, therapeutic antibodies have increasingly been shown to be effective in the treatment of a variety of diseases. Existing vaccines, such as measles vaccine, polio vaccine, hepatitis b vaccine, and hepatitis a vaccine, all provide neutralizing antibodies to vaccinees to prevent viral infection. Since neutralizing antibodies can destroy viruses before they enter cells and can scavenge free viruses outside cells in vivo, neutralizing antibodies in the blood of patients who recover from viral infection can be used for the treatment of viral infections.

Disclosure of Invention

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

The first object of the present invention is to provide a neutralizing antibody against SARS-COV-2, a severe acute respiratory syndrome type II coronavirus, 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 more combinations 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.

The second objective of the invention is to provide a gene encoding the neutralizing antibody.

The third purpose of the invention is to provide an expression vector carrying the gene.

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

The fifth purpose of the invention is to provide the application of the neutralizing antibody in preparing a medicament for treating pneumonia COVID-19.

The sixth purpose of the invention is to provide a kit for treating pneumonia COVID-19, wherein the kit contains the neutralizing antibody.

The invention has the beneficial effects that:

the invention extracts peripheral immune cells from blood of a patient recovering from COVID-19, screens B cells which can be combined with a new coronavirus antigen protein-spike protein from the peripheral immune cells, and analyzes single B cells for producing antibodies at a single cell level to obtain gene sequences which encode heavy chains and light chains of variable regions of neutralizing antibodies in the B cells. The sequences can be used for reconstructing and expressing a neutralizing antibody 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 diagram of a procedure for screening for neutralizing antibodies using individual B cells in the blood of a convalescent patient with COVID-19;

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

FIG. 3 shows a negative control antibody Pmab (no neutralizing effect) and a neutralizing antibody HC5K VH/B5K VL (IC) ₅₀ 0.1273 mug/mL) pseudovirus neutralization experiment result;

FIG. 4 shows the results of neutralization experiments (IC) of the neutralizing antibody D5K VH/B5K VL pseudovirus ₅₀ ～0.00033μg/mL)；

FIG. 5 shows the neutralizing antibody HC5K VH/D4K VL (IC) ₅₀ About 0.00084. Mu.g/mL) and the neutralizing antibody HF4L VH/D5KVL (IC) ₅₀ About 0.0046 mug/mL) pseudovirus neutralization assay results;

FIG. 6 shows the neutralizing antibody HC5K VH/D5K VL (IC) ₅₀ About 0.0066. Mu.g/mL) and neutralizing antibody HD5K VH/A5KVL (IC) ₅₀ About 0.0017. Mu.g/mL) pseudovirus neutralization assay results;

FIG. 7 shows the neutralizing antibody HF4L VH/A5K VL (IC) ₅₀ About 0.6049. Mu.g/mL) and the neutralizing antibody HF4L VH/B5K VL (IC) ₅₀ About 0.6077 mug/mL) pseudovirus neutralization experiment result;

FIG. 8 shows the neutralizing antibody HD5K VH/D4K VL (IC) ₅₀ 0.1996. Mu.g/mL) pseudovirus neutralization assay results.

Detailed Description

The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can carry out the present invention, but the embodiments are not to be construed as limiting the present invention.

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

About 15mL of blood of a plurality of patients with the new coronavirus pneumonia is extracted, peripheral Blood Mononuclear Cells (PBMC) in the blood of each patient are separated and extracted by a Ficoll gradient method, and the obtained PBMC are washed twice for standby.

Fc block is added into each PBMC sample, 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 are added in turn. Followed by addition of Streptavidin-APC and action for 30 min. Antibody-labeled PBMC samples were then collected using FACS AriaTMIII flow cytometer loading analysis. 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, two antigens marked by Biotin can be specifically combined with Streptavidin marked by APC, so that the APC positive cells are cells capable of being specifically combined with SARS-COV-2. Selecting CD3 ^- CD19 ⁺ CD27 ⁺ APC ⁺ The cells in (1) are memory B cells which are searched for and can be specifically combined with SARS-COV-2. Separating single B cell with flow cytometer, pumping into 96-well plate with cell lysate, sealing film, freezing on dry ice, and storing at-80 deg.c. Thus, a single B cell capable of specifically binding to SARS-COV-2 can be isolated.

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

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

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

RT-PCR amplification of single B cell antibody light and heavy chain variable region gene

RT-PCR primers (SEQ ID NO. 9-25) were designed as shown in Table 1:

TABLE 1

Single B cell RT-PCR was performed using the selected single B cell as a template. The PCR system configuration and sample adding are completed in a biological safety cabinet and are operated on ice, and an RT-PCR reaction system and reaction conditions are as follows:

and (3) PCR reaction system:

TABLE 2

Composition (I)	System of	Final concentration
			RNase-free water	-	-
5 × RT-PCR buffer	10.0μl	1×
			dNTP mixture	2.0μl	dNTP 400. Mu.M each
Primer A	-	0.6μM
			B primer	-	0.6μM
Mixed enzyme	2.0μl
			RNase inhibitors	-	5-10 units
Template RNA	-	1pg–2μg
			General System	50.0μl	-

And (3) PCR reaction conditions:

nest type PCR amplification single B cell antibody light and heavy chain variable region gene

Nested PCR primers (SEQ ID NO. 26-38) were designed as shown in Table 3:

TABLE 3

Using RT-PCR products as templates to establish three PCR reaction systems, respectively amplifying variable region genes of H chain, kappa chain and lambda chain of the antibody, wherein each reaction system respectively uses a mixed primer corresponding to the reaction system, and the nested PCR reaction system and the reaction conditions are as follows:

and (3) PCR reaction system:

TABLE 4

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

And (3) PCR reaction conditions:

example 3: results of the experiment

The application obtains 5 light chain variable region sequences and 3 heavy chain variable region sequences from recovered patients in B cells capable of combining with the spike protein of the new coronavirus. 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 new coronavirus SARS-COV-2.

Wherein, 5 light chain variable regions are respectively named as 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), and 3 heavy chain variable regions are respectively named as HD5K VH (SEQ ID NO. 6), HF4L VH (SEQ ID NO. 7) and HC5K VH (SEQ ID NO. 8).

Expression and purification of the antibody were performed as follows:

in this example, 9 antibodies were constructed, respectively: 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.

1. Vector construction

Primers were designed to amplify the corresponding VH and VL sequences according to the corresponding antibody light and heavy chains. The amplified VH and VL sequences were recombined onto the backbone plasmid of the antibody expression vector using recombinase (Cloneexpress II One Step Cloning Kit, C112-01/02, vazyme), and the bacterial solution was subjected to sequencing for validation.

2. Plasmid extraction

The correctly confirmed clony was inoculated into LB (Amp 100 ug/ml) medium, cultured overnight at 37 ℃, and the next day, the plasmid was extracted with a plasmid macroextraction kit, and the concentration was measured.

3. Transient expression of cells

The extracted plasmid was transiently transformed into 293F cells as follows:

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

2) 100ug of plasmid was diluted with medium.

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

4) Adding the plasmid/PEI mixture to the cell suspension, gently shaking the flask during the addition, placing at 37 ℃ 8% ₂ Culturing at 125 rpm.

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

6) The supernatant was harvested on day 6.

4. Antibody purification

1) Harvesting the supernatant

And (4) centrifuging by using a centrifuge, and harvesting a supernatant. The supernatant was filtered through a 0.4um filter membrane and the column was connected.

2) Protein purification

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

5. The resulting protein was purified by SDS-PAGE analysis

The purified antibody was analyzed by SDS-PAGE electrophoresis. The concentration of the separating gel used in the analysis is 4-20%. Reducing the sample, adding the reduced sample into a Loading Buffer, boiling for 10min at 70 ℃, and then Loading for analysis. The results are shown in FIG. 2.

Example 4: pseudovirus neutralization assay

This experiment was performed to evaluate new corona neutralizing antibodies against pseudoviruses carrying the spike protein of new corona viruses. The virus system takes HIV-1 carrying luciferase reporter gene as a virus framework, simultaneously expresses new coronavirus Spike protein in a virus shell, forms pseudovirion to infect exogenous cell lines with high expression ACE2, highly simulates the invasion process of the new coronavirus to target cells through Spike-ACE2, and the degree of infection of the target cells by the pseudovirion is positively correlated with the luminous value of luciferase and negatively correlated with the neutralizing activity of antibody.

The neutralizing antibody is combined with S protein of virus shell in vitro, and the site on the S protein combined with ACE2 is sealed, so that the S protein cannot be combined with ACE2, and the virus cannot invade cells; on the contrary, the antibody without neutralization activity cannot interfere the combination of the S protein and ACE2 on the cell surface, the virus entering the cell can express Fluc protein, and the luminous value of the virus is detected by an enzyme-labeling instrument after the virus reacts with a luminous substrate.

The experimental steps are as follows:

1. sample preparation: pre-thawing pseudovirus in refrigerator or ice from-80 deg.C to 4 deg.C, diluting the virus with DMEM medium containing 10% FBS to 1-2X 10 ⁴ TCID ₅₀ mL (if the infected ACE2 overexpressing cell line was purchased by non-self, then the optimal TCID50 needed to be self-learned).

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

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

4. Column 3-10 were each charged with 90. Mu.l serum-free DMEM, then 45. Mu.l of the dilution from column 2 was added to column 3 using a row gun for dilution by multiple fold until column 9 was reached, and the excess 45. Mu.l of the final column was discarded. Then 90. Mu.l of the diluted pseudovirus solution was added to each well. 90. Mu.L of pseudovirus was added to the virus control group (VC), 180. Mu.L of serum-containing DMEM medium was added to the cell control group (CC), and the 96-well cell culture plate was incubated at 37 ℃ for 1 hour.

5. The sample-pseudovirus mixture in 96-well cell culture plates and the two control groups were divided into three transfers to 96-well white plates at 50. Mu.L/well (3 replicates).

6. Immediately after 50. Mu.l of the solution had a density of 0.4X 10 ⁶ cells/ml (2X 10 in number) ⁴ cells/well) were plated into 96-well plates (without medium change) and incubated in an incubator at 37 ℃.

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

8. And continuously culturing for 48h, taking out a 96-hole white plate, balancing to room temperature, adding 125 mu l of room-temperature-balanced Bio-Lite reporter gene detection reagent into each hole, vibrating the plate for 2min, standing at room temperature for 5min, and detecting a chemiluminescence value (RLU) by using an enzyme-labeling instrument.

9. Data processing: log values of antibody concentrations and corresponding RLUs were taken into graph pad software, and IC50 values were calculated and compared.

The results of the experiment are shown in table 5 and fig. 3 to 8: the neutralizing antibody of the invention has certain neutralizing capacity to the new coronavirus. However, the neutralizing capacity of each antibody to the new coronavirus is different. The lower the IC50 value in the virus neutralization experiment, the better, indicating that only few antibodies are needed to neutralize the virus. Neutralizing antibodies against the novel coronaviruses have been reported to have IC50 values in virus neutralization experiments higher than 0.5. Mu.g/mL to 0.0012. Mu.g/mL. In the novel coronavirus neutralizing antibody, the IC50 of some antibodies such as HD5K VH/B5K VL and HC5K VH/D4K VL in a virus neutralizing experiment reaches 0.00033 mu g/mL and 0.00084 mu g/mL, and the neutralizing capacity of the antibody on viruses is stronger than that of most novel coronavirus neutralizing antibodies.

TABLE 5 neutralizing antibody virus neutralizing ability

Antibodies	Virus neutralization assay 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-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention, such as base substitution of less than 3% for the heavy chain or light chain sequence of the antibody variable region, or amino acid substitution of less than 3% for the amino acid sequence of the portion of the antibody expressed by the heavy chain or light chain sequence of the antibody variable region, are within the scope of the present invention. The protection scope of the invention is subject to the claims.

Sequence listing

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<212> DNA

<213> (Artificial sequence)

<400> 15

gctctgtgac ctcctatgag ctg 23

<210> 16

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 16

ggtctctctc scagcytgtg ctg 23

<210> 17

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 17

gttcttgggc caattttatg ctg 23

<210> 18

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 18

ggtccaattc ycaggctgtg gtg 23

<210> 19

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 19

gagtggattc tcagactgtg gtg 23

<210> 20

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 20

caccagtgtg gccttgttgg cttg 24

<210> 21

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 21

acaggtgccc actcccaggt gcag 24

<210> 22

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 22

aaggtgtcca gtgtgargtg cag 23

<210> 23

<211> 27

<212> DNA

<213> (Artificial sequence)

<400> 23

cccagatggg tcctgtccca ggtgcag 27

<210> 24

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 24

caaggagtct gttccgaggt gcag 24

<210> 25

<211> 22

<212> DNA

<213> (Artificial sequence)

<400> 25

tcttgtccac cttggtgttg ct 22

<210> 26

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 26

atgacccagw ctccabycwc cctg 24

<210> 27

<211> 22

<212> DNA

<213> (Artificial sequence)

<400> 27

gtgctgtcct tgctgtcctg ct 22

<210> 28

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 28

ctgctaccgg ttcctgggcc cagtctgtgc tgackcag 38

<210> 29

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 29

ctgctaccgg ttcctgggcc cagtctgccc tgactcag 38

<210> 30

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 30

ctgctaccgg ttctgtgacc tcctatgagc tgacwcag 38

<210> 31

<211> 37

<212> DNA

<213> (Artificial sequence)

<400> 31

ctgctaccgg ttctctctcs cagcytgtgc tgactca 37

<210> 32

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 32

ctgctaccgg ttcttgggcc aattttatgc tgactcag 38

<210> 33

<211> 38

<212> DNA

<213> (Artificial sequence)

<400> 33

ctgctaccgg ttccaattcy cagrctgtgg tgacycag 38

<210> 34

<211> 28

<212> DNA

<213> (Artificial sequence)

<400> 34

ctcctcactc gagggyggga acagagtg 28

<210> 35

<211> 18

<212> DNA

<213> (Artificial sequence)

<400> 35

sargtgcagc tcgtggag 18

<210> 36

<211> 18

<212> DNA

<213> (Artificial sequence)

<400> 36

gaggtgcagc tgttggag 18

<210> 37

<211> 20

<212> DNA

<213> (Artificial sequence)

<400> 37

ggaattctca caggagacga 20

<210> 38

<211> 25

<212> DNA

<213> (Artificial sequence)

<400> 38

agtagtcctt gaccaggcag cccag 25

Claims

1. A neutralizing antibody against SARS-COV-2, a severe acute respiratory syndrome type II coronavirus 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.2;

the nucleotide sequence of the heavy chain variable region DNA sequence is SEQ ID NO.6.

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 neutralising antibody as claimed in claim 1 or claim 2 in the manufacture of a medicament for the treatment of pneumonia COVID-19.

7. A kit for treating pneumonia COVID-19, comprising the neutralizing antibody of claim 1 or 2.