Monoclonal neutralizing antibody for resisting respiratory syncytial virus and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a monoclonal neutralizing antibody for resisting respiratory syncytial virus and application of the monoclonal neutralizing antibody. The antibody provided by the invention can identify non-denatured RSV (L) protein through ELISA (enzyme-linked immunosorbent assay), but cannot identify denatured RSV (D) protein, and is a kappa IgG2b type antibody. The molecular marker is 0.0001 mu g / ml-1 mu g / ml, can positively recognize the RSV Pre-F protein, and has a gradient effect. The antibody can be used as a neutralizing active antibody in an ELISA double-antibody sandwich method, can be used for detecting the in-vitro potency of the RSV PreF protein, and plays an important role in evaluating the vaccine activity in the vaccine research and development process.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly relates to a monoclonal neutralizing antibody against respiratory syncytial virus and its application. Background Art

[0002] Respiratory syncytial virus (RSV) is a common respiratory virus that mainly infects infants, the elderly, and people with weakened immune systems.

[0003] Respiratory syncytial virus belongs to the Paramyxoviridae family and is a single-stranded negative-sense RNA virus. There are two antigenic types, A and B, both of which are prevalent. The attachment protein (G) and fusion protein (F) on the surface of RSV are the main antigens that can induce neutralizing antibody responses. The G protein is responsible for the virus to adhere to the cell membrane surface, but it has antigenic variability. The F protein is responsible for the virus to fuse with the cell, presenting more neutralizing antibody-targeted epitopes. It is the main target of most vaccines and immunotherapeutic drugs under development. The F protein has two conformations, pre-fusion and post-fusion, and most strong neutralizing epitopes are concentrated in the pre-fusion conformation of the F protein.

[0004] Therefore, it is necessary to develop antibodies that are specific and have neutralizing activity against the pre-fusion conformation of the F protein. Summary of the Invention

[0005] The present invention uses RSV Pre-F antigen to immunize mice with Freund's complete adjuvant, expresses antibodies through hybridoma cells, and finally obtains a monoclonal neutralizing antibody against respiratory syncytial virus with significant effects, thereby completing the present invention.

[0006] The present invention provides a monoclonal neutralizing antibody against respiratory syncytial virus, which comprises at least 1 antibody heavy chain variable region and at least 1 antibody light chain variable region, and wherein the antibody heavy chain variable region has CDRH1, CDRH2, and CDRH3 with amino acid sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5: The antibody light chain variable region has CDRL1, CDRL2, and CDRL3, wherein the amino acid sequence of CDRL1 is as shown in SEQ ID NO: 8, the amino acid sequence of CDRL2 is YTS, and the amino acid sequence of CDR3 is as shown in SEQ ID NO: 9.

[0007] Specifically, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 7.

[0008] The present invention also provides a coding gene for the monoclonal neutralizing antibody against respiratory syncytial virus.

[0009] More specifically, the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO: 6.

[0010] The present invention also provides the use of the monoclonal neutralizing antibody against respiratory syncytial virus in the preparation of RSV Pre-F protein or an ELISA detection reagent or kit for detecting respiratory syncytial virus.

[0011] Specifically, it is used for the evaluation of the vaccine activity of respiratory syncytial virus.

[0012] The present invention provides an ELISA detection kit for detecting RSV Pre-F protein or respiratory syncytial virus, using the monoclonal neutralizing antibody against respiratory syncytial virus as a neutralizing activity antibody and carrying a detectable label.

[0013] Furthermore, the kit further includes a second antibody carrying a detectable label, and the second antibody is another anti-RSV antibody.

[0014] Specifically, the detectable label is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0015] The present invention also provides the use of the monoclonal neutralizing antibody against respiratory syncytial virus in the preparation of a drug for preventing and / or treating diseases related to respiratory syncytial virus infection.

[0016] The antibody 18E7 of the present invention can ELISA recognize the non-denatured RSV (L) protein, but cannot recognize the denatured RSV (D) protein, indicating that 18E7 is a monoclonal antibody recognizing the conformational epitope of the antigen and is a κIgG2b type antibody. It can positively recognize the RSV Pre-F protein between 0.0001 μg / ml and 1 μg / ml and has a gradient effect. Using it as a neutralizing activity antibody in the ELISA double antibody sandwich method can be used to detect the in vitro potency of the RSV Pre-F protein, which plays an important role in the evaluation of vaccine activity during the vaccine R & D process. Description of the Drawings

[0017] Figure 1 The standard curve of the ELISA detection in Example 6. Detailed Embodiments

[0018] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0019] Unless otherwise specified, the experimental materials, reagents, and instruments used in the examples of the present invention are all commercially available. Unless otherwise specified, the technical means used in the examples are all conventional means well known to those skilled in the art.

[0020] Example 1. Preparation of hybridoma cell lines of monoclonal antibodies 1. Animal immunization 1) Basic immunization: RSVPre-F antigen and Freund's complete adjuvant were mixed in equal volumes and fully emulsified, and injected subcutaneously at several points. A total of 2 mice were immunized, and the injection volume for each Balb / c mouse was 50 μg each time.

[0021] 2) Booster immunization: Booster immunization uses an emulsion of antigen and Freund's incomplete adjuvant. Three days before cell fusion, a physiological saline solution containing 15 μg of antigen is injected intraperitoneally. The experimental results are shown in Table 1.

[0022] Table 1. Detection of immune mouse serum (OD450)

[0023] From the results in Table 1, it can be seen that the serum titer of mouse No. 1 was the highest, and this mouse was selected for spleen extraction, fusion with SP20 cells and subsequent preparation of hybridoma cells.

[0024] 2. Preparation of hybridoma cells According to the conventional method, the spleen cells of mice were collected and fused with SP2 / 0 cells at a ratio of 10:1 with 500g / L PEG4000. The cells were selected and cultured with HAT semi-solid culture medium (IMDM culture medium containing 1-3% methylcellulose, 1*HAT, and 10-20% fetal bovine serum) for 10-20 days, and 1000 clones were picked and cultured in liquid DMEM medium in 96-well plates. The culture was continued for 5-10 days, and when the cells grew up, half of the culture medium was aspirated for initial screening of clones by indirect ELISA.

[0025] The operation steps of ELISA method are as follows: use 200ng / well of RSV Pre-F protein to coat the plate, use immune mouse serum 1:2000 as positive control, culture supernatant without clone growth as negative control, add 100μl of 1:2000 HRP-goat anti-mouse IgG to each well, and finally measure the 450nm OD value. Any OD450 value greater than 2 times of the negative control can be preliminarily determined as a positive clone.

[0026] Table 2. Primary Screening Positive Clones (OD450)

[0027] Select the primary screening positive clones, transfer them to a 24-well plate for continued culture for 5 - 10 days. After the cells grow, aspirate the cell culture medium from each well and perform indirect ELISA to rescreen the clones. The results of the rescreening are shown in Table 3 below.

[0028] Table 3. Summary Table of Rescreening Clone Results (OD450)

[0029] Table 3 is a summary table of all clones with OD450 values greater than twice the OD450 value of the negative control during rescreening. The rescreening results of the remaining primary screening positive clones are negative. Therefore, only the rescreening positive clones are retained for subsequent research.

[0030] 3. Ascites Preparation and Antibody Purification Select adult BALB / c mice and inoculate pristane intraperitoneally at 0.5 ml per mouse. After 7 - 10 days, inoculate hybridoma cells intraperitoneally at 1×10 6 -2×10 6 per mouse. After a 5-day interval, when the abdomen is significantly enlarged and the skin feels tense when touched by hand, 3.5 ml of ascites can be collected using a 16-gauge needle.

[0031] The amount of ascites collected is at a reasonable level for each clone. Centrifuge the ascites (13000 r / min for 30 minutes) to remove cell components and other precipitates, and collect the supernatant. Purify it using Protein G - Sepharose CL-4B. The loading buffer is 20 mM PBS buffer, and the column chromatography elution buffer is: glycine buffer at pH 2.7, 20 mM, to obtain the monoclonal antibody against RSV.

[0032] Example 2. Detection of Antibody Neutralizing Activity In a 96-well cell culture plate, add 100 μL of Hep-2 cells (1.0×10 4 cells / well) to each well and culture overnight in an incubator at 37°C and 5% CO 2 Dilute the test serum 40-fold as the first dilution and then perform 4-fold serial dilutions. Repeat for 2 wells for each dilution, with 60 μL per well; according to the virus titration results, dilute the virus to 1500 TCID50 / 100 μL with MEM complete medium and inoculate 60 μL per well. Incubate in an incubator at 37°C and 5% CO 2 for 1 hour, then transfer 100 μL / well to a 96-well cell culture plate and place it in an incubator at 37°C and 5% CO 2Cultivate overnight in an incubator. Set up virus control wells and cell control wells. After cultivation, take out the cell culture plate from the incubator and place it in a cell imaging microplate reader to read the number of fluorescent cells expressed in each well. Use the Reed-Muench method to calculate the dilution multiple corresponding to 50% fluorescence inhibition in the sample wells, which is the neutralizing antibody titer value of this serum.

[0033] Table 4. IC50 of different clones

[0034] Note: NA means the inhibition rate cannot reach IC50, that is, it is a non-neutralizing antibody.

[0035] From the results in Table 4, it can be seen that the neutralizing activity sensitivity of the 18E7 clone is much higher than that of the other clones, and the sensitivity is more than ten times that of other clones. Therefore, the 18E7 clone was selected for further research.

[0036] Example 3. Identification of the 18E7 antibody 1. Identification of the conformational epitope of the antibody Treatment with denatured protein RSV (D): Incubate non-denatured protein RSV (L) with 0.2M sodium carbonate, 0.01M DTT, pH 10.6 at room temperature for 30 minutes and then boil for 5 minutes. Coating: Dilute the RSV Pre-F protein to 1 μg / ml, add 100 μl to a 96-well microplate, and coat overnight at 4°C. Pour out the coating solution, wash twice with PBST, pat dry, and then add 200 μL of 2% bovine serum albumin (BSA) to each well, and incubate in a 37°C incubator for 1.5 hours for blocking. Add 100 μL of the antibody to each well and incubate in a 37°C incubator for 1.5 hours for blocking. After washing the plate, add 100 μL / well of goat anti-mouse secondary antibody, incubate at 37°C for 1 hour, after washing and patting dry, add the chromogenic agent for color development, incubate at room temperature for 15 min, add 50 μL / well of the stop solution, and read the absorbance at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Table 5. Detection of conformational epitope

[0037] The results in Table 5 show that 18E7 can recognize the non-denatured RSV (L) protein by ELISA, but cannot recognize the denatured RSV (D) protein, indicating that 18E7 is a monoclonal antibody that recognizes the conformational epitope of the antigen.

[0038] 2. Subtype identification of the antibody Using the indirect ELISA method, use antibodies against various IgG2b subtypes of mice to identify the IgG2b subtype of the antibody produced by the above hybridoma cells. The results are shown in Table 6 below.

[0039] Table 6. Subtype identification of the 18E7 antibody

[0040] The results in Table 6 show that the 18E7 antibody is a κIgG2b type antibody.

[0041] Example 4. Detection of the binding activity of the 18E7 antibody Dilute the RSV Pre-F protein to 1 μg / ml, add 100 μl to the ELISA plate of a 96-well plate, and coat overnight at 4°C. Pour out the coating solution, wash twice with PBST, pat dry, then add 200 μL of 2% bovine serum albumin (BSA) to each well, and incubate in a 37°C incubator for 1.5 hours for blocking. Add 100 μL of the target 18E7 monoclonal antibody with gradient dilution to each well, and incubate in a 37°C incubator for 1.5 hours for blocking. After washing the plate, add 100 μL / well of goat anti-mouse secondary antibody, incubate at 37°C for 1 hour, after washing and patting dry, add the chromogenic agent for color development, incubate at room temperature for 15 min, add 50 μL / well of the stop solution, and read the absorbance at a wavelength of 450 nm with an ELISA reader.

[0042] Table 7. Binding activity of the 18E7 antibody

[0043] The results are shown in Table 7. The results indicate that 18E7 can positively recognize the RSV Pre-F protein between 0.0001 μg / ml and 1 μg / ml, and has a gradient effect.

[0044] Example 5. Determination of the variable region sequences of the 18E7 clone Extract mRNA from the obtained 18E7 monoclonal cells respectively, reverse transcribe it into cDNA, perform high-fidelity PCR amplification using universal variable region primers, insert the PCR product fragments into the T vector for DNA sequence determination, and translate the obtained sequences into the amino acid sequences of proteins. After aligning the obtained sequences, no identical sequences were shown, indicating that the obtained sequences are specific sequences. The antibody-related sequences of the two clones are as follows: The nucleotide sequence of the variable region of the 18E7 heavy chain is as follows (357 bp): GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTCCTTACTCATTCACTGGCTACTACATGCACTGGGTGAAGCAAAGCCATGTAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTTACAATGCTGCTACTCGCTACAACCAGAATTTCAAGGACAAGGCCAGCTTGACTGTAGATAAGTCCTCCAGCACAGTCTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCGACTATGGTAACTACTTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 1).

[0045] The amino acid sequence encoded by the variable region of the heavy chain of 18E7 is as follows (119aa): EVQLQQSGPELVKPGASVKISCKASPYSFTGYYMHWVKQSHVKSLEWIGRINPYNAATRYNQNFKDKASLTVDKSSSTVYMELHSLTSEDSAVYYCARSDYGNYFFDYWGQGTTLTVSS (SEQ ID NO: 2).

[0046] By identification using conventional methods, it can be known that in the heavy chain: the CDR1 sequence is: PYSFTGYY (SEQ ID NO: 3); the CDR2 sequence is: INPYNAAT (SEQ ID NO: 4); the CDR3 sequence is: ARSDYGNYFFDY (SEQ ID NO: 5).

[0047] The nucleotide sequence of the variable region of the light chain of 18E7 is as follows (318bp): GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTCAATTACATGTACTGGTACCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCAGTTTACTAGTTCCCCGTCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 6).

[0048] The amino acid sequence encoded by the variable region of the 18E7 light chain is as follows (106 aa): ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPSTFGGGTKLEIK (SEQ ID NO: 7).

[0049] By identification according to conventional methods, it can be known that in the light chain: the CDR1 sequence is: SSVNY (SEQ ID NO: 8); the CDR2 sequence is: YTS; the CDR3 sequence is: QQFTSSPST (SEQ ID NO: 9).

[0050] Using the sequences identified above, through known antibody engineering techniques, various genetically engineered antibodies can be prepared, such as chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, etc., and retain the biological characteristics of the monoclonal neutralizing antibody from which they are derived.

[0051] Example 6. Preparation of an ELISA detection reagent for RSV Pre-F protein using the 18E7 antibody Detection method: The coated antibody 18E7 was diluted to 2 μg / mL with 0.05 mol / L carbonate buffer solution at pH 9.6. 100 μL was added to each well of the ELISA plate and coated overnight at 4°C. The coating solution was poured off, and the plate was washed 2 times with PBST, patted dry, and then 200 μL of 3% bovine serum albumin (BSA) was added to each well and incubated in a 37°C incubator for 1.5 hours for blocking. Gradient-diluted standard proteins (200, 100, 50, 25, 12.5, 6.25 ng / ml) were added, 100 μL to each well of the ELISA plate, and incubated in a 37°C incubator for 1.5 hours for blocking. A commercially available anti-RSV antibody [B1537M] (ab252748) was labeled with horseradish peroxidase to obtain B1537M-HRP and stored. After washing the plate, 100 μL / well of B1537M-HRP (0.5 μg / ml) was added and incubated at 37°C for 1 hour. After washing and patting dry, the chromogenic agent was added for color development, incubated at room temperature for 15 min, 50 μL / well of the stop solution was added, and the absorbance was read at 450 nm using an ELISA reader. The four-parameter fitting method was used to logarithmically process the data, and the standard curve was as shown in Figure 1 shown.

[0052] Figure 1 The results of Figure 1 showed that between 6.25 ng / ml and 200 ng / ml, the gradient was obvious, and the R value of the standard curve was greater than 0.99, meeting the requirements. Since one of the antibodies paired in this ELISA double antibody sandwich method is the neutralizing active antibody 18E7, this detection method can be used to detect the in vitro potency of RSV Pre-F protein, which plays an important role in the evaluation of vaccine activity during the vaccine R & D process.

Claims

1. A monoclonal neutralizing antibody against respiratory syncytial virus, characterized in that: The invention comprises at least one antibody heavy chain variable region and at least one antibody light chain variable region, wherein the antibody heavy chain variable region has CDRH1, CDRH2 and CDRH3 with amino acid sequences as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5: The antibody light chain variable region has CDRL1, CDRL2 and CDRL3, wherein the amino acid sequence of CDRL1 is shown in SEQ ID NO: 8, the amino acid sequence of CDRL2 is YTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

9.

2. The monoclonal neutralizing antibody against respiratory syncytial virus according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7.

3. A gene encoding a monoclonal neutralizing antibody against respiratory syncytial virus as claimed in claim 1 or 2.

4. The coding gene according to claim 3, characterized in that The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 1, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:

6.

5. Use of the anti-respiratory syncytial virus monoclonal neutralizing antibody according to claim 1 or 2 in the preparation of RSVPre-F protein or an ELISA detection reagent or kit for detecting respiratory syncytial virus.

6. The use according to claim 5, characterized in that It is used to evaluate the vaccine activity of respiratory syncytial virus.

7. An ELISA detection kit for detecting RSVPre-F protein or respiratory syncytial virus, characterized in that: The anti-respiratory syncytial virus monoclonal neutralizing antibody as claimed in claim 1 or 2 is used as the neutralizing active antibody and is provided with a detectable marker.

8. The kit according to claim 7, characterized in that The kit also includes a second antibody with a detectable label, which is another anti-RSV antibody.

9. The kit according to claim 7, characterized in that The detectable label is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

10. Use of the anti-RSV monoclonal neutralizing antibody according to claim 1 or 2 in the preparation of a drug for preventing and / or treating diseases related to RSV infection.

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