A monoclonal neutralizing antibody against varicella-zoster virus and its application

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses a monoclonal neutralizing antibody against varicella-zoster virus and its application. The antibody provided by the present invention comprises at least one antibody heavy chain variable region and at least one 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 CDR1 with amino acid sequence as shown in SEQ ID NO: 8, CDR2 with amino acid sequence AAT and CDR3 with amino acid sequence as shown in SEQ ID NO: 9. Using it as a detection antibody to construct an ELISA double antibody sandwich method can be used to detect the in vitro efficacy of VZV gE protein, which plays an important role in the evaluation of 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 varicella-zoster virus and an application thereof. Background Art

[0002] Herpes zoster (shingles) is an acute infectious skin disease caused by the varicella-zoster virus (VZV). Its incidence increases significantly with age. Currently, vaccination is the only effective means of prevention and control. Varicella-zoster virus (VZV) is a highly complex virus that co-expresses multiple glycoproteins (e.g., gE, gB, gH, gI, etc.). VZV glycoproteins play important roles in viral infection, transmission, and immune response. Among them, gE is the most critical glycoprotein and is widely used in vaccine development and diagnostic research.

[0003] During vaccine development, the double antibody sandwich method can be used to test in vitro efficacy. Therefore, monoclonal antibodies are an important tool for vaccine antigen quality control, especially antibodies with specificity and neutralizing activity, which play an irreplaceable role in vaccine development. Summary of the Invention

[0004] The present invention uses VZV gE antigen and Freund's complete adjuvant to immunize mice, expresses antibodies through hybridoma cells, and ultimately obtains monoclonal neutralizing antibodies against varicella-zoster virus with significant effects, thereby completing the present invention.

[0005] The present invention provides a monoclonal neutralizing antibody against varicella-zoster 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:

[0006] The antibody light chain variable region has a CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, a CDR2 with an amino acid sequence of AAT, and a CDRL3 with an amino acid sequence as shown in SEQ ID NO: 9.

[0007] 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.

[0008] The present invention also provides a gene encoding the monoclonal neutralizing antibody against varicella-zoster virus.

[0009] 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.

[0010] The present invention also provides the use of the monoclonal neutralizing antibody against varicella-zoster virus in preparing an ELISA detection reagent or a kit for detecting varicella-zoster virus.

[0011] Specifically, it is used for the evaluation of vaccine activity against varicella-zoster virus.

[0012] The present invention particularly provides an ELISA detection kit for detecting varicella-zoster virus, which uses the monoclonal neutralizing antibody against varicella-zoster virus as a detection antibody and is provided with a detectable label.

[0013] Specifically, the detectable label is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.

[0014] Furthermore, the kit further comprises another antibody with a detectable label, and the other antibody is an anti-VZV antibody.

[0015] The present invention also provides use of the monoclonal neutralizing antibody against varicella-zoster virus in the preparation of drugs for preventing and / or treating VZV-related diseases.

[0016] The antibody provided by the present invention can positively recognize VZV gE protein at concentrations between 0.00001 μg / ml and 1 μg / ml, with a gradient effect. Using it as a detection antibody in a double-antibody sandwich ELISA assay can be used to detect the in vitro potency of VZV gE protein. It exhibits neutralizing activity, which is crucial for evaluating vaccine activity during vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the standard curve for detecting VZV gE protein using the ELISA double-antibody sandwich method. DETAILED DESCRIPTION

[0018] 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.

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

[0020] Example 1. Development of hybridoma cell lines producing monoclonal antibodies

[0021] 1. Animal immunization

[0022] 1) Primary immunization: VZV gE antigen and Freund's complete adjuvant were mixed in equal volumes and fully emulsified, and then injected subcutaneously at several sites. A total of 3 mice were immunized, with each Balb / c mouse receiving 50 μg of the injection each time.

[0023] 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.

[0024] Table 1. Detection of immune mouse serum (OD450)

[0025]

[0026] The results in Table 1 show that the serum titer of mouse #2 was the highest, and this mouse was selected for spleen extraction, fusion with SP20 cells, and subsequent preparation of hybridoma cells.

[0027] 2. Preparation of hybridoma cells

[0028] 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.

[0029] The ELISA procedure is as follows: Coat the plate with 200 ng / well of VZV gE protein. Use immune mouse serum at a 1:2000 dilution as a positive control and culture supernatant from a culture medium without clone 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.

[0030] Table 2. Initial screening of positive clones (OD450)

[0031]

[0032] 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.

[0033] Table 3. Summary of clone screening results (OD450)

[0034]

[0035] 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 remaining clones that were positive in the initial screening were negative in the rescreening. Therefore, only the clones that were positive in the rescreening were retained for subsequent studies.

[0036] 3. Ascites Preparation and Antibody Purification

[0037] 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.

[0038] The amount of ascites collected was sufficient 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-VZV monoclonal antibodies were obtained using Protein G-Sepharose CL-4B, with the column loading solution being 20 mM PBS buffer and the column elution solution being 20 mM glycine buffer, pH 2.7.

[0039] Example 2: Neutralizing activity detection of antibodies

[0040] Using the plaque reduction assay, VZV virus is diluted to an appropriate concentration and mixed with equal amounts of each antibody at 20 μg / ml. The mixture is incubated at 37°C for 1-2 hours, and plaque formation is measured. A plaque reduction rate of 50% or greater is considered neutralizing activity for the mAb.

[0041] Table 4

[0042]

[0043] From the results in Table 4, we can see that only the 1E8 clone has an 80% reduction rate of missed shifts, which is significantly greater than 50%. This indicates that the mAb has neutralizing activity, and this clone was selected for further study.

[0044] Example 3: Identification of 1E8 Antibody

[0045] 1. Identification of antibody conformational epitopes

[0046] Denatured VZV (D) protein treatment: Incubate the non-denatured VZV (L) protein with 0.2M sodium carbonate, 0.01M DTT, pH 10.6, at room temperature for 30 minutes, then boil for 5 minutes. Coating: Dilute VZV gE protein to 1μg / ml, add 100μl to a 96-well microtiter plate, and coat overnight at 4°C. Pour off the coating solution, wash twice with PBST, pat dry, then add 200μl of 2% bovine serum albumin (BSA) to each well and block in a 37°C incubator for 1.5 hours. Add 100μl 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 patting dry, add color developer 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.

[0047] Table 5. Conformational epitope detection

[0048]

[0049] The results in Table 5 indicate that 1E8 can recognize non-denatured VZV (L) protein by ELISA, but cannot recognize denatured VZV (D) protein, indicating that 1E8 is a monoclonal antibody that recognizes a conformational epitope of the antigen.

[0050] 2. Identification of Antibody Subtypes

[0051] The IgG subtypes of 1E8 were identified using indirect ELISA using antibodies raised against various mouse IgG subtypes. The results are shown in Table 6 below.

[0052] Table 6. Identification of subtypes of 1E8 antibody

[0053]

[0054] The results in Table 6 indicate that the 1E8 antibody is a κ IgG1 type antibody.

[0055] Example 4: Binding activity detection of 1E8 antibody

[0056] Dilute VZV gE protein to 1 μg / ml, add 100 μl to a 96-well microplate and coat overnight at 4°C. Pour off 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 the target 1E8 monoclonal antibody with serial dilutions 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 colorimetric reagent for color development, incubate at room temperature for 15 minutes, add 50 μl / well of stop solution, and read at a wavelength of 450 nm using a microplate reader.

[0057] Table 7. Binding activity of 1E8 antibody

[0058]

[0059] The results in Table 7 indicate that 1E8 can positively recognize VZV gE protein at concentrations ranging from 0.00001 μg / ml to 1 μg / ml, and has a gradient effect.

[0060] Example 5: Determination of variable region sequence of 1E8 clone

[0061] mRNA was extracted from 1E8 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:

[0062] The nucleotide sequence of the 1E8 heavy chain variable region is as follows (357 bp):

[0063] GAGGTCAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAATCTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGCTCGGCAGGCTCCAGGGAAAGGGCAGGAATGGATTGGAGAAATTAATCCAGGAAGCAGTACGATAAACTAT ACGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACTAGATGGTTACGACAATGCTATGGACTGCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 1).

[0064] The amino acid sequence encoded by the 1E8 heavy chain variable region is as follows (119 aa):

[0065] EVKLLESGGGLVQPGGSLNLSCAASGFDFSRYWMSWARQAPGKGQEWIGEINPGSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARLDGYDNAMDCWGQGTSVTVSS (SEQ ID NO: 2).

[0066] Identification by conventional methods revealed that in the heavy chain, the CDR1 sequence was GFDFSRYW (SEQ ID NO: 3); the CDR2 sequence was INPGSSTI (SEQ ID NO: 4); and the CDR3 sequence was ARLDGYDNAMDC (SEQ ID NO: 5).

[0067] The nucleotide sequence of the 1E8 light chain variable region is as follows (321 bp):

[0068] GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTAATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAACAAAGTTA GCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA (SEQ ID NO: 6).

[0069] The amino acid sequence encoded by the 1E8 light chain variable region is as follows (107 aa):

[0070] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATKLADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPYTFGGGTKLEIK (SEQ ID NO: 7).

[0071] Identification by conventional methods revealed that in the light chain, the CDR1 sequence was ENIYSN (SEQ ID NO: 8); the CDR2 sequence was AAT; and the CDR3 sequence was QHFWGTPYT (SEQ ID NO: 9).

[0072] 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 antibodies from which they are derived.

[0073] Example 6: Preparation of VZV gE ELISA Detection Reagent Using 1E8 Antibody

[0074] Detection Method: Anti-VZV gE protein antibody [#9] (ab272686) 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 (160, 80, 40, 20, 10, and 5 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. The 1E8 antibody was labeled with horseradish peroxidase to produce 1E8-HRP, which was then stored. After washing, 100 μL / well of 1E8-HRP (0.5 μg / ml) was added and incubated at 37°C for 1 hour. After washing and patting dry, a colorimetric reagent was added for color development. The plate was incubated at room temperature for 15 minutes. 50 μL / well of the stop solution was added and the plate was read at a wavelength of 450 nm using a microplate reader. The data were logarithmically processed using the four-parameter fitting method. The resulting standard curve is shown below. Figure 1 shown.

[0075] Figure 1 The results showed a clear gradient between 5 ng / ml and 160 ng / ml, and the R value of the standard curve was greater than 0.99, meeting the requirements. Because one of the antibodies in this ELISA double-antibody sandwich pair is the neutralizing antibody 1E8, this assay can be used to measure the in vitro potency of VZV gE protein, which plays an important role in evaluating vaccine activity during vaccine development.

Claims

1. A monoclonal neutralizing antibody against varicella-zoster 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 has a CDR1 with an amino acid sequence as shown in SEQ ID NO: 3, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and a CDRH3 with an amino acid sequence as shown in SEQ ID NO: 5: The antibody light chain variable region has a CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, a CDR2 with an amino acid sequence of AAT, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:

9.

2. The anti-varicella-zoster virus monoclonal neutralizing antibody 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. The gene encoding the monoclonal neutralizing antibody against varicella-zoster 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 anti-varicella-zoster virus monoclonal neutralizing antibody according to claim 1 or 2 in the preparation of an ELISA detection reagent or kit for detecting varicella-zoster virus.

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

7. An ELISA test kit for detecting varicella-zoster virus, characterized in that: The anti-varicella-zoster virus monoclonal neutralizing antibody according to claim 1 or 2 is used as the detection antibody and is labeled with a detectable marker.

8. 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.

9. The kit according to claim 7, wherein The kit further includes another antibody with a detectable label, wherein the other antibody is an anti-VZV antibody.

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