A monoclonal neutralizing antibody against respiratory syncytial virus and its application

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses a monoclonal neutralizing antibody against respiratory syncytial virus and its application. The antibody provided by the present invention can recognize non-denatured RSV (L) protein by ELISA, but cannot recognize denatured RSV (D) protein, and is a κIgG2b type antibody. It can positively recognize RSV Pre-F protein at concentrations between 0.0001 μg / ml and 1 μg / ml, and has a gradient effect. It is used as a neutralizing antibody in the ELISA double antibody sandwich method and can be used to detect the in vitro efficacy of RSV Pre-F protein, which plays an important role in evaluating vaccine activity during vaccine development.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a monoclonal neutralizing antibody against respiratory syncytial virus and an application thereof. 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 with two antigenic types, A and B, both of which are prevalent. The adsorption 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 the protein responsible for the adhesion of the virus to the cell membrane surface, but it has antigenic variability. The F protein is the protein responsible for the fusion of the virus and the cell, presenting more neutralizing antibody targeting epitopes, and is the main target of most vaccines and immunotherapy drugs currently under development. The F protein has two conformations, pre-fusion and post-fusion. Most strong neutralizing epitopes are concentrated in the F protein in the pre-fusion conformation.

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

[0005] The present invention uses RSVPre-F antigen and Freund's complete adjuvant to immunize mice, expresses antibodies through hybridoma cells, and ultimately obtains monoclonal neutralizing antibodies 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, comprising 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 the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5:

[0007] The antibody light chain variable region comprises 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.

[0008] Specifically, 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.

[0009] The present invention also provides a gene encoding the monoclonal neutralizing antibody against respiratory syncytial virus.

[0010] More specifically, 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.

[0011] The present invention also provides the use of the monoclonal neutralizing antibody against respiratory syncytial virus in preparing RSVPre-F protein or in an ELISA detection reagent or kit for detecting respiratory syncytial virus.

[0012] Specifically, it is used for vaccine activity evaluation against respiratory syncytial virus.

[0013] The invention provides an ELISA detection kit for detecting RSVPre-F protein or respiratory syncytial virus. The kit uses the monoclonal neutralizing antibody against respiratory syncytial virus as a neutralizing active antibody and is provided with a detectable marker.

[0014] Furthermore, the kit also includes a second antibody with a detectable label, and the second antibody is another anti-RSV antibody.

[0015] Specifically, the detectable label is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0016] 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.

[0017] The antibody 18E7 provided herein can recognize the non-denatured RSV (L) protein by ELISA, but not the denatured RSV (D) protein. This indicates that 18E7 is a monoclonal antibody that recognizes a conformational epitope of the antigen and is a κ IgG2b antibody. It positively recognizes the RSV Pre-F protein at concentrations ranging from 0.0001 μg / ml to 1 μg / ml, with a gradient effect. It can be used as a neutralizing antibody in a double-antibody sandwich ELISA assay to assess the in vitro potency of the RSV Pre-F protein, which plays an important role in evaluating vaccine activity during vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , the standard curve of ELISA detection in Example 6. DETAILED DESCRIPTION

[0019] The following examples are provided 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.

[0020] 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 conventional means well known to those skilled in the art.

[0021] Example 1. Development of hybridoma cell lines producing monoclonal antibodies

[0022] 1. Animal immunization

[0023] 1) Primary immunization: RSVPre-F antigen was mixed with Freund's complete adjuvant in equal volumes and fully emulsified, and then injected subcutaneously at different points. A total of 2 mice were immunized, with each Balb / c mouse receiving 50 μg of the vaccine per injection.

[0024] 2) Booster Immunization: Booster immunization used an emulsion of antigen and Freund's incomplete adjuvant. Three days before cell fusion, 15 μg of antigen in saline solution was injected intraperitoneally. The experimental results are shown in Table 1.

[0025] Table 1. Detection of immune mouse serum (OD450)

[0026]

[0027] 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 removal, fusion with SP20 cells, and subsequent preparation of hybridoma cells.

[0028] 2. Preparation of Hybridoma Cells

[0029] Mouse spleen cells were harvested as per conventional methods and fused with SP2 / 0 cells at a ratio of 10:1 using 500 g / L PEG4000. Cells were selected and cultured in HAT semisolid medium (IMDM containing 1-3% methylcellulose, 1% HAT, and 10-20% fetal bovine serum) for 10-20 days. 1000 clones were then transferred to a 96-well plate and cultured in liquid DMEM medium. Culture was continued for 5-10 days. Once the cells had grown, half of the culture medium was aspirated and used for initial screening of clones using indirect ELISA.

[0030] The ELISA procedure is as follows: Coat the plate with 200 ng / well of RSV Pre-F protein. Use immune mouse serum at a 1:2000 dilution as a positive control and culture supernatant from a culture medium lacking colony growth as a negative control. Add 100 μl of HRP-goat anti-mouse IgG at a 1:2000 dilution to each well and measure the OD value at 450 nm. Any OD450 value at least twice that of the negative control is considered a positive clone.

[0031] Table 2. Initial screening of positive clones (OD450)

[0032]

[0033] The clones that were positive in the initial screening were selected and transferred to 24-well plates for further culture. After 5 to 10 days of culture, the cell culture medium from each well was aspirated for indirect ELISA and the clones were rescreened. The results of the rescreening are shown in Table 3.

[0034] Table 3. Summary of clone screening results (OD450)

[0035]

[0036] Table 3 is a summary of all clones whose OD450 values ​​were greater than 2 times the OD450 value of the negative control during rescreening. The rescreening results of the remaining clones that were positive in the initial screening were negative. Therefore, only the clones that were positive in the rescreening were retained for subsequent studies.

[0037] 3. Ascites Preparation and Antibody Purification

[0038] Adult BALB / c mice were selected and intraperitoneally inoculated with pristane (0.5 ml per mouse). 7-10 days later, hybridoma cells were intraperitoneally inoculated (1×10 cells per mouse). 6 -2×10 6 After an interval of 5 days, when the abdomen is noticeably enlarged and the skin feels tense when touched, 3.5 ml of ascites can be collected using a 16-gauge needle.

[0039] The amount of ascites collected was at an appropriate level for each clone. The ascites was centrifuged (13,000 rpm for 30 minutes) to remove cellular components and other precipitates, and the supernatant was collected. Anti-RSV monoclonal antibodies were obtained using Protein G-Sepharose CL-4B, with the column loading solution being 20 mM PBS buffer and the column eluent being 20 mM glycine buffer, pH 2.7.

[0040] Example 2: Neutralizing activity detection of antibodies

[0041] In a 96-well cell culture plate, 100 μL of Hep-2 cells (1.0×10 4Each well was incubated overnight at 37°C with 5% CO2. The test serum was diluted 40-fold, followed by four-fold serial dilutions, with duplicate wells at each dilution, using 60 μL per well. Based on the viral titration results, the virus was diluted to 1500 TCID50 / 100 μL in MEM complete medium, and 60 μL was inoculated into each well. After incubation for 1 hour in a 37°C with 5% CO2 incubator, 100 μL / well was transferred to a 96-well cell culture plate and incubated overnight at 37°C with 5% CO2. Virus and cell control wells were also set up. After incubation, the cell culture plate was removed from the incubator and placed on a cell imaging plate reader to read the number of fluorescent cells in each well. The neutralizing antibody titer of the serum was calculated using the Reed-Muench method at the dilution factor that resulted in 50% inhibition of fluorescence in the sample well.

[0042] Table 4. IC50 of different clones

[0043]

[0044] Note: NA means the inhibition rate does not reach IC50, which means it is a non-neutralizing antibody.

[0045] From the results in Table 4 , it can be seen that the neutralization activity sensitivity of clone 18E7 is much higher than that of the other clones, and the sensitivity is more than ten times that of the other clones, so clone 18E7 was selected for further study.

[0046] Example 3: Identification of 18E7 Antibody

[0047] 1. Identification of antibody conformational epitopes

[0048] Denatured RSV protein (D) treatment: Incubate the non-denatured RSV protein (L) with 0.2M sodium carbonate, 0.01M DTT, pH 10.6, at room temperature for 30 minutes, then boil for 5 minutes. Coating: Dilute RSVPre-F protein to 1 μg / ml, and add 100 μl to a 96-well microtiter plate. Coat overnight at 4°C. Discard the coating solution, wash twice with PBST, pat dry, then add 200 μl of 2% bovine serum albumin (BSA) to each well and block in a 37°C incubator for 1.5 hours. Add 100 μl of antibody to each well and block in a 37°C incubator for 1.5 hours. After washing, add 100 μl / well of goat anti-mouse secondary antibody and incubate at 37°C for 1 hour. After washing and pat dry, add color development reagent and incubate at room temperature for 15 minutes. Add 50 μl / well of stop solution, and read using a microtiter plate reader at 450 nm.

[0049] Table 5. Conformational epitope detection

[0050]

[0051] The results in Table 5 show that 18E7 can recognize non-denatured RSV (L) protein by ELISA, but cannot recognize denatured RSV (D) protein, indicating that 18E7 is a monoclonal antibody that recognizes a conformational epitope of the antigen.

[0052] 2. Identification of Antibody Subtypes

[0053] The IgG2b subtype of the antibodies produced by the hybridoma cells was identified by indirect ELISA using antibodies against various mouse IgG2b subtypes. The results are shown in Table 6 below.

[0054] Table 6. Identification of subtypes of 18E7 antibody

[0055]

[0056] The results in Table 6 indicate that the 18E7 antibody is a kappa IgG2b type antibody.

[0057] Example 4: Binding activity detection of 18E7 antibody

[0058] RSV Pre-F protein was diluted to 1 μg / ml, and 100 μl was added to a 96-well microplate and coated overnight at 4°C. The coating solution was discarded, the plates were washed twice with PBST, and patted dry. Then, 200 μl of 2% bovine serum albumin (BSA) was added to each well and the plates were blocked in a 37°C incubator for 1.5 hours. 100 μl of the target 18E7 monoclonal antibody with serial dilutions was added to each well and the plates were blocked in a 37°C incubator for 1.5 hours. After washing, 100 μl / well of goat anti-mouse secondary antibody was added to the plates and the plates were incubated at 37°C for 1 hour. After washing and patting dry, the colorimetric reagent was added for color development, incubated at room temperature for 15 minutes, 50 μl / well of stop solution was added, and the plates were read at a wavelength of 450 nm using a microplate reader.

[0059] Table 7. Binding activity of 18E7 antibody

[0060]

[0061] The results are shown in Table 7. The results showed that 18E7 could positively recognize RSVPre-F protein at concentrations ranging from 0.0001 μg / ml to 1 μg / ml, and had a gradient effect.

[0062] Example 5: Determination of variable region sequences of 18E7 clones

[0063] mRNA was extracted from each of the 18E7 monoclonal cells, reverse transcribed into cDNA, and amplified using high-fidelity PCR using universal primers for the variable regions. The PCR product fragments were inserted into a T-vector for DNA sequencing and translated into protein amino acid sequences. Comparison of the obtained sequences revealed no identical sequences, indicating that the obtained sequences were specific. The relevant antibody sequences of the two clones are as follows:

[0064] The nucleotide sequence of the 18E7 heavy chain variable region is as follows (357 bp):

[0065] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCCCTTACTCATTCACTGGCTACTACATGCACTGGGTGAAGCAAAGCCATGTAAAGAGCCTTGAGTGGATTGGACGTATTAATCCTTACAATGCTGCTACTCGCTAC AACCAGAATTTCAAGGACAAGGCCAGCTTGACTGTAGATAAGTCCTCCAGCACAGTCTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCGACTATGGTAACTACTTCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 1).

[0066] The amino acid sequence encoded by the 18E7 heavy chain variable region is as follows (119aa):

[0067] EVQLQQSGPELVKPGASVKISCKASPYSFTGYYMHWVKQSHVKSLEWIGRINPYNAATRYNQNFKDKASLTVDKSSSTVYMELHSLTSEDSAVYYCARSDYGNYFFDYWGQGTTLTVSS (SEQ ID NO: 2).

[0068] Identification by conventional methods revealed that in the heavy chain, the CDR1 sequence was PYSFTGYY (SEQ ID NO: 3); the CDR2 sequence was INPYNAAT (SEQ ID NO: 4); and the CDR3 sequence was ARSDYGNYFFDY (SEQ ID NO: 5).

[0069] The nucleotide sequence of the 18E7 light chain variable region is as follows (318 bp):

[0070] GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTCAATTACATGTACTGGTACCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACACATCCAACCTGGC TCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCAGTTTTACTAGTTCCCCGTCCACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA (SEQ ID NO: 6).

[0071] The amino acid sequence encoded by the light chain variable region of 18E7 is as follows (106 aa):

[0072] ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFTSSPSTFGGGTKLEIK (SEQ ID NO: 7).

[0073] Identification by conventional methods revealed that in the light chain, the CDR1 sequence was SSVNY (SEQ ID NO: 8); the CDR2 sequence was YTS; and the CDR3 sequence was QQFTSSPST (SEQ ID NO: 9).

[0074] Using the above-identified sequences, various genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, single-chain antibodies, and diabodies, can be prepared through known antibody engineering techniques, while retaining the biological properties of the monoclonal neutralizing antibodies from which they are derived.

[0075] Example 6: Preparation of ELISA Detection Reagent for RSVPre-F Protein Using 18E7 Antibody

[0076] Detection Method: Coating antibody 18E7 was diluted to 2 μg / mL in 0.05 mol / L carbonate buffer (pH 9.6). 100 μL was added to each well of an ELISA plate and coated overnight at 4°C. The coating solution was decanted, the plates were washed twice with PBST, and patted dry. Then, 200 μL of 3% bovine serum albumin (BSA) was added to each well and the plates were blocked in a 37°C incubator for 1.5 hours. A serial dilution of a standard protein (200, 100, 50, 25, 12.5, and 6.25 ng / mL) was added, 100 μL was added to each well of the plate, and the plates were blocked in a 37°C incubator for 1.5 hours. A commercially available anti-RSV antibody [B1537M] (ab252748) was labeled with horseradish peroxidase to generate B1537M-HRP and stored. After washing, add 100 μL / well of B1537M-HRP (0.5 μg / ml) and incubate at 37°C for 1 hour. After washing and patting dry, add colorimetric reagent for color development and incubate at room temperature for 15 minutes. Add 50 μL / well of stop solution and read the plate at a wavelength of 450 nm using a microplate reader. Using the four-parameter fitting method, logarithmize the data and obtain the standard curve as shown below. Figure 1 shown.

[0077] Figure 1 The results showed a clear gradient between 6.25 ng / ml and 200 ng / ml, with an R value of greater than 0.99, meeting the requirements. Because one of the antibodies in this ELISA double-antibody sandwich pair is the neutralizing antibody 18E7, this assay can be used to measure the in vitro potency of RSV Pre-F protein, which plays an important role in evaluating vaccine activity during vaccine development.

Claims

1. A monoclonal neutralizing antibody against respiratory syncytial virus, characterized in that Comprising at least one antibody heavy chain variable region and at least one antibody light chain variable region; The antibody heavy chain variable region comprises CDRH1, CDRH2 and CDRH3, wherein the amino acid sequence of CDRH1 is shown in SEQ ID NO: 3, the amino acid sequence of CDRH2 is shown in SEQ ID NO: 4, and the amino acid sequence of CDRH3 is shown in SEQ ID NO: 5: The antibody light chain variable region comprises 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 CDRL3 is shown in SEQ ID NO:

9.

2. The monoclonal neutralizing antibody against respiratory syncytial virus according to claim 1, wherein 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. The gene encoding the monoclonal neutralizing antibody against respiratory syncytial virus according to claim 1 or 2.

4. The coding gene according to claim 3, wherein 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 monoclonal neutralizing antibody against respiratory syncytial virus according to claim 1 or 2 in the preparation of 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 kit for detecting RSVPre-F protein or respiratory syncytial virus, characterized in that: The anti-respiratory syncytial virus monoclonal neutralizing antibody according to claim 1 or 2 is used as the neutralizing active antibody and is labeled with a detectable marker.

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

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

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