CN119331085B

CN119331085B - Sabei coronavirus broad-spectrum neutralizing antibodies and their applications

Info

Publication number: CN119331085B
Application number: CN202411442675.1A
Authority: CN
Inventors: 江力玮; 左腾; 李玲; 陈希贤; 杜锐萍; 王作伟
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2024-10-16
Filing date: 2024-10-16
Publication date: 2025-03-14
Anticipated expiration: 2044-10-16
Also published as: CN119331085A

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to a broad-spectrum neutralizing antibody of a sand Bei Guan rhabdovirus and application thereof. The antibody is any one or a combination of a plurality of KXD350, KXD352, KXD355 and KXD358, wherein each of the KXD350, KXD352, KXD355 and KXD358 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has three heavy chain CDRs, and the light chain variable region has three light chain CDRs. The 4 broad-spectrum neutralizing antibodies and the variable region sequences of the heavy chain and the light chain disclosed by the invention provide a basis for constructing the Sha Beiguan-shaped virus therapeutic neutralizing antibody with high affinity, high quality and strong specificity, and have higher application value for prevention, control and treatment of coronaviruses.

Description

Sha Beiguan-like virus broad-spectrum neutralizing antibody and application thereof

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a broad-spectrum neutralizing antibody for a sand Bei Guan-like virus and application thereof.

Technical Field

Coronaviruses can be divided into four genera, α, β, γ, δ, where coronaviruses of both genera α and β primarily infect mammals, and all infectious human coronaviruses are also located in both genera. The beta genus coronavirus is divided into 4 independent subgroups A, B, C and D lineage, of which B lineage is also called Sha Beiguan coronavirus (Sarbecovirus), and only one virus species SARS-related coronavirus, namely SARS-associated coronavirus, including SARS-CoV-1, SARS-CoV-2 and variants thereof, and some animal derived SARS-associated coronaviruses (Lan,Q.,et al.,Pan-coronavirus fusion inhibitors to combat COVID-19and other emerging coronavirus infectious diseases.J Med Virol,2023.95(1):p.e28143.).SARS-CoV-1 are representative members of Sha Beiguan coronaviruses, and recently the outbreak of epidemic SARS-CoV-2 coronavirus is newly identified Sha Beiguan coronavirus.

The coronavirus genome mainly encodes four structural proteins spike (S), nucleocapsid (N), membrane (M) and envelope (E) proteins as well as non-structural proteins, which serve to maintain the structural integrity of the enveloped virion. Coronavirus S proteins form trimers on the viral surface that are able to bind to ACE2 surface receptors on host cells and mediate the entry of coronaviruses into host cells. It is therefore the primary target for therapeutic neutralizing antibodies, and is also the focus of drug development and vaccine design.

A total of 7 human-infected coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV-2, MERS-CoV) are currently found. In the 21 st century, coronaviruses SARS-CoV-1, MERS-CoV and SARS-CoV-2 have successively exploded, and have been pandemic many times worldwide, causing acute respiratory infections, and constitute a great threat to public health, and also indicate the possibility of future re-outbreaks of highly pathogenic coronaviruses.

Therefore, there is a need to develop broad-spectrum neutralizing antibodies against the sand Bei Guan-like virus as a technical reserve to cope with the potential outbreak potential of the future coronaviruses.

Disclosure of Invention

In order to solve the problems in the prior art, it is an object of the present invention to provide a broad-spectrum neutralizing antibody for the arenavirus Bei Guan.

The invention adopts the following technical scheme:

sha Beiguan a broad spectrum neutralizing antibody, said neutralizing antibody being a combination of any one or more of KXD350, KXD352, KXD355 and KXD358, each of said KXD350, KXD352, KXD355 and KXD358 comprising a heavy chain variable region having three heavy chain CDRs and a light chain variable region having three light chain CDRs, wherein:

The three CDR amino acid sequences of the KXD350 heavy chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 in sequence, and the three CDR amino acid sequences of the KXD350 light chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO.6 in sequence;

The three CDR amino acid sequences of the KXD352 heavy chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 in sequence, and the three CDR amino acid sequences of the KXD352 light chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 in sequence;

The three CDR amino acid sequences of the KXD355 heavy chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 in sequence, and the three CDR amino acid sequences of the KXD355 light chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18 in sequence;

The three CDR amino acid sequences of the KXD358 heavy chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21 in sequence, and the three CDR amino acid sequences of the KXD358 light chain variable region are amino acid sequences with homology of 80% or more with the amino acid sequences shown in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24 in sequence.

The above-mentioned homologous sequence is preferably an amino acid sequence having a homology of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The homologous sequence also comprises an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence shown in the sequence listing.

Preferably, the 6 CDR amino acid sequences of the KXD350 heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;

The 6 CDR amino acid sequences of the KXD352 heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12;

The 6 CDR amino acid sequences of the KXD355 heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO.16, SEQ ID NO. 17 and SEQ ID NO. 18;

the 6 CDR amino acid sequences of KXD358 heavy chain variable region and light chain variable region are shown as SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, respectively.

Specifically, the CDR amino acid sequences of the heavy chain variable region and the light chain variable region of KXD350, KXD352, KXD355, KXD358 are shown in the following table:

In the table, H represents a heavy chain and K represents a light chain.

Preferably, the amino acid sequence of the heavy chain variable region of KXD350 is shown as SEQ ID NO. 25, the amino acid sequence of the light chain variable region of KXD350 is shown as SEQ ID NO. 26, the amino acid sequence of the heavy chain variable region of KXD352 is shown as SEQ ID NO. 27, the amino acid sequence of the light chain variable region of KXD352 is shown as SEQ ID NO. 28, the amino acid sequence of the heavy chain variable region of KXD355 is shown as SEQ ID NO. 29, the amino acid sequence of the light chain variable region of KXD355 is shown as SEQ ID NO. 30, the amino acid sequence of the heavy chain variable region of KXD358 is shown as SEQ ID NO. 31, and the amino acid sequence of the light chain variable region of KXD358 is shown as SEQ ID NO. 32.

Preferably, the neutralizing antibody further comprises an Fc domain.

Preferably, the Fc domain is a human IgG Fc domain, including a human IgG1 Fc domain, an IgG2Fc domain, an IgG3 Fc domain, or an IgG4 Fc domain.

Preferably, the neutralizing antibody further comprises an immunological marker coupled thereto, wherein the immunological marker is any one of an enzyme marker, a fluorescein marker, an isotope marker and a biotin marker.

The present invention also provides a polynucleotide encoding a KXD350, KXD352, KXD355 or KXD358 neutralizing antibody as described above, or encoding a neutralizing antibody comprising an Fc domain as described above;

Wherein the nucleotide sequence of the KXD350 heavy chain variable region is shown as SEQ ID NO. 33, the nucleotide sequence of the KXD350 light chain variable region is shown as SEQ ID NO. 34, the nucleotide sequence of the KXD352 heavy chain variable region is shown as SEQ ID NO. 35, the nucleotide sequence of the KXD352 light chain variable region is shown as SEQ ID NO. 36, the nucleotide sequence of the KXD355 heavy chain variable region is shown as SEQ ID NO. 37, the nucleotide sequence of the KXD355 light chain variable region is shown as SEQ ID NO. 38, the nucleotide sequence of the KXD358 heavy chain variable region is shown as SEQ ID NO. 39, and the nucleotide sequence of the KXD358 light chain variable region is shown as SEQ ID NO. 40.

The invention also provides an expression vector comprising a polynucleotide as described above, and a host cell comprising an expression vector as described above. Preferably, the host cell is a host cell for expressing a foreign protein, e.g., a bacterium, yeast, insect cell, mammalian cell.

The invention provides application of the neutralizing antibody in preparing a medicament for treating and/or preventing the infection of the sand Bei Guan-like virus.

Preferably, the Sha Beiguan-like viruses include SARS-CoV-1 and SARS-CoV-2 and variants thereof, including Delta, BA.1, BA.4/5, BA.2.75, BQ.1, XBB.1.5, EG.5.1, HK.3, BA.2.86, JN.1, KP.2, KP.3.

The present invention provides a pharmaceutical composition comprising any one or a combination of at least two neutralizing antibodies as described above, further comprising a pharmaceutically acceptable carrier.

The invention provides application of the neutralizing antibody in preparation of a kit for detecting the sand Bei Guan-like virus.

The invention provides a kit for detecting the sand Bei Guan rhabdovirus, which comprises the neutralizing antibody.

The invention provides the use of a neutralizing antibody as described above in the preparation of a vaccine for the prevention of a sand Bei Guan-like viral infection.

The invention also provides a vaccine comprising a neutralising antibody as described above.

The invention has the beneficial effects that:

The invention screens 4 groups of antibodies KXD350, KXD352, KXD355 and KXD358 with strong broad-spectrum neutralization activity on Sha Beiguan-1 SARS-CoV-2 and variants thereof, including the latest epidemic variants EG.5.1, HK.3, JN.1, KP.2 and KP.3 of SARS-CoV-2, clones out light chain and heavy chain variable regions thereof, obtains nucleotide sequences thereof after sequencing, provides a basis for constructing Sha Beiguan-like virus therapeutic neutralization antibodies with high affinity, high quality and strong specificity, and has higher application value on prevention, control and treatment of Sha Beiguan-like viruses.

Drawings

FIG. 1 is an antigen-specific flow sort of single B cells;

FIG. 2 is ELISA readings of SARS-CoV-2WT Spike and SARS-CoV-1Spike double positive antibody supernatant;

FIG. 3 shows the neutralization IC ₅₀ values of the 4-strain antibodies against various pseudoviruses.

Detailed Description

For easy understanding, the following description will make more specific use of the technical solution of the present invention in conjunction with the examples:

Example 1

Discovery and preparation of antibodies

1.1 Acquisition of antigen

SARS-CoV-2WT Spike extracellular region (1-1208) and SARS-CoV-1Spike extracellular region (1-1195) were constructed with TRIMER TAG of bacteriophage T-4 fibritin and 8 XHis, tain-STREPIITAG, flag tag at the C-terminus, respectively, on vector pCAGGS to form expression plasmids. The plasmid and a transfection reagent PEI are gently mixed, incubated for 20min at room temperature, and 293F suspension cells are transfected to obtain recombinant cells, and protein expression is carried out. The recombinant cells were cultured in SMM 293-TII complete medium for 96 hours, then centrifuged at 3000rpm for 30min at 4℃and the supernatant was collected. Purifying by Strep-Tactin magnetic bead affinity chromatography to obtain target protein WT Spike. Specific purification procedure 100ml of supernatant was mixed with Strep-Tactin magnetic beads, incubated overnight at 4℃and the solution and beads were magnetically separated. Adding 30ml PBS solution into the magnetic beads, mixing under rotation, washing the magnetic beads for 5min, collecting the magnetic beads by using a magnetic rack, discarding supernatant, and repeating the washing step three times. To the beads 10ml of elution buffer (5 mM D-Desthiobiotin in PBS) was added, mixed by tumbling at room temperature and incubated for 15min. The magnetic beads were collected using a magnetic separation rack and the eluate containing the protein of interest was transferred to a clean centrifuge tube. The concentrate was performed using an ultrafiltration tube with a 100kDa cut-off and the solution was replaced with PBS buffer.

1.2 Antigen-specific flow sorting of Single B cells

1 SARS-CoV-2Omicron breakthrough infected was recruited as volunteers. Before infection, volunteers were vaccinated with two needles of mRNA vaccine in 2021 and infected with omacron variants for the first time in 2022, the dominant strains now popular were BA.5 and BF.7, 5ml of peripheral blood was withdrawn after 9 months of omacron breakthrough recovery, plasma and blood cells were obtained by centrifugation, and peripheral blood mononuclear cell PBMC was obtained by density gradient centrifugation using lymphocyte separation medium (Ficoll-Paque PLUS density gradient centrifugation medium, sizhuan, cat# 17144002). The PBMCs were flow stained and antigen-specifically sorted using a flow cytometer. Flow sorting and loop gate strategy DAPI ^-CD3^-CD19⁺CD27⁺IgD^- and sorting was performed on His and Flag double positive memory B cells and plasmablasts of the antigen SARS-CoV-2WT Spike, single B cells were sorted into 96 well plates containing cell lysates as shown in fig. 1.

1.3 Antibody variable region Gene amplification

Reverse transcription of the cleaved mRNA to cDNA is performed using single B cell gene amplification techniques, and the gene products of the heavy and light chain variable regions are obtained by PCR using single B cell antibody gene amplification primers and sequenced. The sequences were analyzed using IgBlast tool.

1.4 Construction of recombinant plasmids

The heavy chain variable region DNA molecule is seamlessly cloned to an expression vector hIgG, and a heavy chain expression plasmid is obtained.

The light chain variable region DNA molecule is seamlessly cloned to an expression vector hIgkappa or hIglambda, and a light chain expression plasmid is obtained.

The coding regions of both the heavy and light chains consist of three parts, a signal peptide, a variable region and a constant region.

1.5 Screening of antibodies

Finally cloning successfully and pairing to obtain the polyclonal antibody. And co-transfecting HEK-293T cells with the paired heavy chain expression plasmid and light chain expression plasmid, culturing for 48 hours, and expressing the antibody to obtain a cultured antibody supernatant. Antibody supernatants were used for primary screening for antibody affinity and neutralization activity.

1.5.1ELISA binding experiments screening for Positive clones

1) Antigen coating the SARS-CoV-2WT Spike antigen and the SARS-CoV-1Spike antigen were diluted in PBS to a final concentration of 1 ng/. Mu.l. ELISA plates were coated 100 μl per well at 4℃overnight.

2) Blocking, discard solution, wash 3 times with 200 μl PBST per well. 200 μl of blocking solution was added to each well and blocked at 37℃for 2h.

3) Washing, the supernatant was discarded, and 200. Mu.l of PBST was added to each well to wash 3 times, and the wells were then patted dry.

4) Cell culture antibody supernatant was added, 3-fold gradient dilution of antibody supernatant, 100 μl of antibody solution was added per well, the first well was undiluted stock solution, and the negative control was PBS. Incubate for 1h at 37 ℃.

5) Adding secondary antibody, discarding supernatant, adding 200 μl of PBST into each well, cleaning for 3 times, and drying. Mu.l of secondary antibody with HRP-labeled goat anti-human IgG (H+L) was added to each well. Incubate for 1h at 37 ℃.

6) Color development, removing supernatant, adding 200 μl of PBST into each well, washing for 4 times, and drying. After 100. Mu.l of the color-developing solution TMB was added and the color was changed, 50. Mu.l of 10% sulfuric acid stop solution was added to each well to terminate the reaction.

7) Reading, namely reading absorbance at 450nm of the enzyme label instrument. Each cell culture antibody supernatant was provided with 1 set of duplicate wells, and each set of experiments was independently repeated 1 time. And selecting the absorbance average value higher than 2.0 as a positive antibody.

As shown in Table 1, it can be seen that the cell culture antibody supernatant was subjected to preliminary screening of ELISA binding experiments to obtain 28 strains of double positive antibodies against SARS-CoV-2WT Spike and SARS-CoV-1Spike, and FIG. 2 shows ELISA readings of the antibody supernatant binding antigens SARS-CoV-2WT Spike and SARS-CoV-1 Spike.

1.5.2 Antibody neutralization Activity Primary screening

The antibody supernatant of the 28 ELISA double positive cell culture was further subjected to neutralization activity screening against SARS-CoV-2WT and SARS-CoV-1 pseudoviruses.

1) Preparation of pseudoviruses

The SARS-CoV-2WT and SARS-CoV-1 pseudoviruses were thawed at room temperature from-80℃and the relative titer of the pseudoviruses was adjusted to about 10 ten thousand (the reading value of the luciferase reporter gene).

2) Taking 96-well cell culture plates, adding 200 μl PBS to the periphery of the 96-well plates, adding 100 μl of culture medium to each of the rest wells, respectively adding 50 μl of antibody supernatant of different cell cultures to the first and fifth columns, mixing, taking 50 μl to the next column Kong Hun, performing gradient dilution by 3 times, diluting 4 gradients altogether, wherein the dilution of the first well is 4.5 times, adding 50 μl of SARS-CoV-2 pseudovirus solution to each well, and standing and incubating in a 37 ℃ cell culture box for 1 hour. The last two columns are cell control and virus control, respectively. An equal volume of PBS buffer was used instead of antibody supernatant as a virus control. An equal volume of DMEM medium containing 10% fetal bovine serum was used as a cell control instead of sham virus solution.

3) The above cell culture plates were taken and 50. Mu.l of ACE2-HEK293T cell suspension was added to each of the middle 60 wells, 2X 10 ⁴ ACE2-HEK293T cells per well, and the culture was continued for 48 hours in a 37℃cell incubator.

4) Taking the cell culture plate, discarding the supernatant, adding 100 μl PBS and 20 μl luciferase reporter gene detection reagent into each well, standing at room temperature in dark place, and incubating for 2min.

5) Taking the cell culture plate, and detecting the luciferase activity.

Each antibody supernatant was provided with 1 set of multiplex wells. Neutralization experiments were independently repeated 2 times.

Neutralization activity (%) = [1- (fluorescence intensity of test group-fluorescence intensity of cell control)/(fluorescence intensity of virus control-fluorescence intensity of cell control) ] ×100%.

The experimental results are shown in table 1.

The antibody supernatant of the cell culture was defined as having a neutralization activity of >80% against SARS-CoV-2WT pseudovirus at 4.5-fold dilution, and a neutralization activity of >60% against SARS-CoV-1 pseudovirus was defined as having a broad-spectrum neutralization activity, and 4 strains were designated as KXD350, KXD352, KXD355 and KXD358, respectively.

TABLE 1ELISA binding and pseudovirus neutralization Activity antibody screening

1.6 Preparation of antibodies

1.6.1 Cotransfecting 293F cells with the heavy chain expression plasmid and the light chain expression plasmid of the 4 antibodies obtained by the above screening to obtain recombinant cells. Culturing in SMM 293-TII complete medium for 96h, then centrifuging at 4 ℃ and 3000rpm for 30min, and collecting supernatant.

1.6.2Protein A magnetic bead affinity chromatography purification of antibodies

The affinity chromatography packing comprises Protein A magnetic beads, an elution buffer solution, a neutralization buffer solution and a neutralization buffer solution, wherein the elution buffer solution comprises 0.1M glycine and the pH value is 3.0, and the neutralization buffer solution comprises 1M Tris and the pH value is 8.5.

50Ml of the supernatant was mixed with Protein A magnetic beads, incubated at 4℃for 16h, and the solution and beads were separated by a magnetic rack. Adding 20ml PBS solution into the magnetic beads, mixing under rotation, washing the magnetic beads for 5min, collecting the magnetic beads by using a magnetic rack, discarding supernatant, and repeating the washing step three times. 9ml of elution buffer is added to the magnetic beads, the mixture is quickly resuspended, mixed uniformly, turned over and mixed at room temperature, and incubated for 5min. Magnetic beads were collected using a magnetic separation rack and the supernatant containing the eluted antibody was transferred to a clean centrifuge tube. To 9ml of eluate, 1ml of neutralization buffer was immediately added to neutralize the pH in order to maintain the biological activity of the antibody and avoid inactivation of the antibody.

1.6.3 Solution Displacement

The antibody solution obtained in the step 2 was concentrated by an ultrafiltration tube, and the solution was replaced with PBS buffer to obtain an antibody solution having a protein concentration of 1 mg/ml.

Example 2

Broad spectrum neutralization test

Preparation of pseudoviruses of SARS-CoV-1 and SARS-CoV-2 and variants thereof

The method comprises the following steps:

S1, artificially synthesized SARS-CoV-1Spike and SARS-CoV-2WT Spike and variants thereof Spike, including Delta, BA.1, BA.4/5, BA.2.75, BQ.1, XBB.1.5, EG.5.1, HK.3, BA.2.86, JN.1, KP.2, KP.3 genes were cloned into pcDNA3.1 expression vectors.

S2, co-transfecting HEK-293T cells with the recombinant plasmid pcDNA3.1/Spike, the plasmid pLenti-CMV-puro-Lucifease and the plasmid psPAX to obtain recombinant cells.

Packaging plasmid psPAX is a plasmid capable of expressing lentiviral capsids, the expression products of which can more readily cross the cell membrane by adhesion mechanisms. The plasmid pLenti-CMV-puro-Luciferase is a lentiviral vector plasmid and the reporter Luciferase gene is expressed in host cells. The envelope glycoprotein in the lentiviral vector is replaced with the novel coronavirus S protein, which results in a pseudovirus that mimics the infection of the novel coronavirus. Pseudoviruses infect target cells through surface S proteins and express reporter luciferase genes.

S3, culturing the recombinant cells obtained in the step S2 in a DMEM medium containing 10% fetal calf serum for 60 hours.

S4, collecting culture supernatant, and centrifuging to obtain supernatant, namely the virus liquid of pseudoviruses of SARS-CoV-1 and SARS-CoV-2 original strains and variant strains.

S5, detecting the virus titer in each pseudo virus liquid by using a luciferase reporter gene detection reagent.

2 Detection of neutralizing Activity of antibodies

The neutralizing antibody can block the binding of the Spike protein of the pseudovirus and the receptor ACE2, thereby preventing the infection of host cell ACE2-HEK293T by the pseudovirus. By detecting the expression level of the reporter luciferase, the degree of blocking of the virus can be deduced, and screening or verification of the neutralizing antibody can be performed.

The test antibody solutions were antibody solutions KXD350, KXD352, KXD355 and KXD358 prepared in example 1, respectively.

The method comprises the following steps:

S1, preparation of antibodies and pseudoviruses

The purified monoclonal antibodies and each pseudovirus solution were removed from-80 ℃ and thawed at room temperature, the relative titer of pseudoviruses was adjusted to about 10 ten thousand (reading of luciferase reporter), the antibodies were filter sterilized and the concentration was adjusted to 0.5mg/ml.

S2, taking a 96-well cell culture plate, adding 200 mu l of PBS to the surrounding wells of the 96-well plate, adding 100 mu l of culture medium to each of the rest wells, supplementing 45.6 mu l of culture medium to each of the first row of wells, adding 4.4 mu l of antibody to each of the wells, uniformly mixing, taking 50 mu l to the next row of wells Kong Hun uniformly, carrying out 3-fold multiple dilution, adding 8 dilutions, and finally adding 50 mu l of pseudovirus liquid to each of the wells. The initial concentration of antibody was 10 ng/. Mu.l, incubated at 37℃for 1 hour in a cell incubator with cell and virus controls in the last two columns, respectively. An equal volume of PBS buffer (pH 7.4, 10 mM) was used instead of the antibody dilution as a virus control. An equal volume of DMEM medium containing 10% fetal bovine serum was used as a cell control in place of SARS-CoV-2 virus solution.

S3, taking out the cell culture plate in the step S2, adding 50 mu lACE of 2-HEK293T cell suspension into the middle 60 holes, and continuously culturing 2X 10 ⁴ ACE2-HEK293T cells in each hole for 48 hours in a 37 ℃ cell culture incubator.

S4, taking out the cell culture plate in the step 3, discarding the supernatant, adding 100 μl PBS and 20 μl luciferase reporter gene detection reagent into each well, standing at room temperature in a dark place, and incubating for 2min.

S5, taking out the cell culture plate in the step 4, and detecting the luciferase activity.

Each antibody was provided with 1 set of duplicate wells. The neutralization experiments were independently repeated 1 time.

The concentration of antibody at 50% neutralization activity, i.e., IC ₅₀ value of antibody, was calculated using Prism 8 software.

The results of neutralization of IC50 values are shown in table 2 and fig. 3.

Table 2 neutralization IC ₅₀ value (ng/. Mu.l) of antibodies against pseudoviruses

As is clear from the neutralization test results, the 4-strain antibodies KXD350, KXD352, KXD355 and KXD358 have small IC ₅₀ values for SARS-CoV-2WT, delta, BA.1, BA.4/5, BA.2.75, BQ.1, XBB.1.5, EG.5.1, HK.3, BA.2.86, JN.1, KP.2, KP.3 pseudoviruses and SARS-CoV-1 pseudoviruses, and have potent broad-spectrum neutralization activities.

Overall, experimental results show that 4 antibodies KXD350, KXD352, KXD355 and KXD358 have strong neutralizing efficacy against SARS-CoV-1 and SARS-CoV-2 and different variants thereof, including the most recently prevalent strains EG.5.1, HK.3, JN.1, KP.2 and KP.3, and have potent broad-spectrum neutralizing activity. Human-infectious coronaviruses among Sha Beiguan are SARS-CoV-1 and SARS-CoV-2 and variants thereof, and thus it is found that these 4 antibodies are potent broad-spectrum neutralizing antibodies against Sha Beiguan and have values for further research and development into therapeutic drugs. The sequences referred to in the antibodies and examples are shown in the sequence listing.

The above embodiments are only for illustrating the technical scheme of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that any modifications, equivalent substitutions and improvements etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Sha Beiguan a broad spectrum neutralizing antibody, wherein said neutralizing antibody is a combination of any one or more of KXD350, KXD352, KXD355, and KXD358, each of said KXD350, KXD352, KXD355, and KXD358 comprising a heavy chain variable region having three heavy chain CDRs and a light chain variable region having three light chain CDRs, wherein:

the three CDR amino acid sequences of the KXD350 heavy chain variable region are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 in sequence, and the three CDR amino acid sequences of the KXD350 light chain variable region are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 in sequence;

The three CDR amino acid sequences of the KXD352 heavy chain variable region are shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 in sequence, and the three CDR amino acid sequences of the KXD352 light chain variable region are shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 in sequence;

The three CDR amino acid sequences of the KXD355 heavy chain variable region are shown as SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 in sequence, and the three CDR amino acid sequences of the KXD355 light chain variable region are shown as SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18 in sequence;

The three CDR amino acid sequences of the KXD358 heavy chain variable region are shown in SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21 in sequence, and the three CDR amino acid sequences of the KXD358 light chain variable region are shown in SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24 in sequence.

2. The Sha Beiguan-like virus broad-spectrum neutralizing antibody according to claim 1, wherein the heavy chain variable region amino acid sequence of KXD350 is shown in SEQ ID NO. 25, the light chain variable region amino acid sequence of KXD350 is shown in SEQ ID NO. 26, the heavy chain variable region amino acid sequence of KXD352 is shown in SEQ ID NO. 27, the light chain variable region amino acid sequence of KXD352 is shown in SEQ ID NO. 28, the heavy chain variable region amino acid sequence of KXD355 is shown in SEQ ID NO. 29, the light chain variable region amino acid sequence of KXD355 is shown in SEQ ID NO. 30, and the light chain variable region amino acid sequence of KXD358 is shown in SEQ ID NO. 31, and the light chain variable region amino acid sequence of KXD358 is shown in SEQ ID NO. 32.

3. The neutralizing antibody of any one of claims 1-2, further comprising an Fc domain.

4. The neutralizing antibody of claim 3 wherein said Fc domain is a human IgG Fc domain.

5. The antibody of claim 3, further comprising an immunological tag coupled to the neutralizing antibody, the immunological tag being any one of an enzymatic tag, a fluorescein tag, an isotope tag, and a biotin tag.

6. A polynucleotide encoding the neutralizing antibody of any one of claims 1-4.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. Use of a neutralizing antibody according to any one of claims 1-4 for the manufacture of a medicament for the treatment and/or prophylaxis of a sal Bei Guan rhabdovirus infection.

10. The use of claim 9, wherein Sha Beiguan rhabdoviruses include SARS-CoV-1 and SARS-CoV-2 and variants thereof including Delta, ba.1, ba.4/5, ba.2.75, bq.1, xbb.1.5, eg.5.1, hk.3, ba.2.86, jn.1, kp.2, kp.3.

11. A pharmaceutical composition comprising the neutralizing antibody of any one of claims 1-4 and a pharmaceutically acceptable carrier.

12. Use of a neutralizing antibody according to any one of claims 1-5 in the preparation of a kit for detecting Sha Beiguan-like viruses.

13. A kit for detecting a sand Bei Guan-like virus comprising a neutralizing antibody according to any one of claims 1-5.

14. Use of a neutralizing antibody according to any one of claims 1-4 for the preparation of a Sha Beiguan-like virus infected vaccine.

15. A vaccine comprising the antibody of any one of claims 1-4.