Poxvirus murine monoclonal antibodies and uses thereof
By preparing a mouse monoclonal antibody specific to the H3L protein, the problem of lacking high-affinity anti-poxvirus antibodies in the existing technology has been solved, achieving specific binding and infection inhibition of poxvirus, and has broad application potential.
Patent Information
- Application Number
- CN202510000072.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-01
- Publication Date
- 2026-01-09
- Estimated Expiration
- 2045-01-01
AI Technical Summary
The lack of high-affinity and highly specific anti-poxvirus antibodies in existing technologies has resulted in insufficient rapid detection and immunotherapy methods for orthopoxviruses such as monkeypoxvirus.
A mouse monoclonal antibody specific to the H3L protein was prepared using hybridoma technology. Balb/c mice were fused with myeloma cells SP2/0, and high-affinity hybridoma cell lines were screened to obtain an antibody with a heavy chain of IgG2b and a light chain of Igκ, which was used to specifically bind to the poxvirus H3L protein and inhibit infection.
The prepared antibody can specifically bind to the H3L protein, significantly inhibiting poxvirus infection. It has broad application value and neutralizing effect, and is suitable for poxvirus detection and immunotherapy.
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Figure CN119735670B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology. Background Technology
[0002] Monkeypox is an infectious disease caused by the monkeypox virus (MPXV), which continues to spread widely worldwide. The monkeypox virus belongs to the orthopoxvirus genus (OPV), which also includes vaccinia virus (VACV), variola virus (VARV), and ectromelia virus (ECTV), among others. These poxviruses share high homology. Monkeypox virus, or other genetically modified poxviruses, can infect humans across species. Because large-scale immunization has ceased for nearly 40 years, most of the population currently lacks immunity to OPV members, including VARV. Therefore, screening for high-affinity, highly specific anti-poxvirus antibodies is essential, providing a strategic antibody reserve for rapid detection of poxviruses and immunotherapy of the disease.
[0003] Orthozvoviral infection of host cells occurs in two forms: mature virions (MV) and extracellular enveloped virions (EVs). The H3L protein, a surface protein shared by all OPV members, contains 324 amino acid residues edited from the H3L gene. It is expressed late in host cell infection, inserts into the MV membrane post-translation, and binds to a hydrophobic transmembrane region near the C-terminus. H3L binds to cell surface molecules and participates in OPV adhesion to host cells, making it a key target in MPXV vaccine design. Summary of the Invention
[0004] This invention uses monkeypox virus H3L protein as an antigen to immunize mice. Hybridoma technology is used to fuse spleen cells from Balb / c mice immunized with H3L protein with myeloma cells SP2 / 0. Following clonal specificity testing, positive clones are selected after three rounds of specificity screening, thus obtaining a hybridoma cell line secreting H3L-specific antibodies. Through cell culture, a murine monoclonal antibody against the H3L protein is obtained. The heavy chain type of this antibody is IgG2b, with the V region belonging to the IgH-V7183 VH5 family; the light chain type is Igκ, with the V region belonging to the IgKV21 family. The antibody type is IgG2b. The function of this antibody is tested, including its binding to H3L and in vitro neutralization experiments.
[0005] The amino acid sequence of the heavy chain variable region of the murine vaccinia virus monoclonal antibody provided by this invention is SEQ ID NO.1, and the heavy chain variable region belongs to the IgH-V7183 VH5 family; the amino acid sequence of the light chain variable region is SEQ ID NO.3, and the light chain variable region belongs to the IgKV21 family.
[0006] In a specific embodiment of the present invention, the constant region sequences of the heavy and light chains of the vaccinia virus murine monoclonal antibody are shown in SEQ ID NO.7 and SEQ ID NO.8.
[0007] In a specific embodiment of the present invention, the heavy chain and light chain amino acid sequences of the vaccinia virus murine monoclonal antibody are as shown in SEQ ID NO.5 and SEQ ID NO.6, indicating that the heavy chain type is IgG2b and the light chain type is Igκ.
[0008] In a specific embodiment of the present invention, the murine monoclonal antibody against poxvirus can specifically bind to the H3L antigen of poxvirus, thereby inhibiting the infection of cells by poxvirus and playing a protective role.
[0009] The beneficial effects of this invention are:
[0010] This invention constructs an H3L protein antigen for specific immunization, uses hybridoma technology to perform cell fusion to prepare an anti-H3L hybridoma cell line, and determines the proliferation and monoclonal antibody production capacity of the H3L hybridoma through clonal detection. This preparation method can be applied to the preparation of other viral antibodies and has broad applicability. Attached Figure Description
[0011] Figure 1 Sequence alignment of poxvirus H3L protein
[0012] Figure 2 SDS-PAGE image of H3L protein expression.
[0013] Figure 3 : Western blot analysis of the light and heavy chains of the antibody.
[0014] Figure 4 Antibody sequence amplification diagram.
[0015] Figure 5 Neutralization experiment diagram. Detailed Implementation
[0016] This invention uses H3L protein as an antigen to immunize mice. Hybridoma cell lines are prepared by fusing spleen cells from Balb / c mice inoculated with H3L antigen with myeloma cells SP2 / 0. The antibody levels in the supernatant of these hybridoma cell lines are then detected to determine their antibody levels and specificity, resulting in a murine monoclonal antibody with poxvirus protection. The function of this antibody is then tested, including its binding to proteins such as H3L and BSA, its binding to viruses such as VCV and ETCV, and in vitro neutralization experiments.
[0017] The amino acid sequence of the heavy chain variable region domain of the vaccinia virus murine monoclonal antibody of the present invention is shown in SEQ ID NO.1 of the sequence listing, and the amino acid sequence of the light chain variable region domain is shown in SEQ ID NO.3 of the sequence listing. The antibody is named H489.
[0018] The amino acid sequence of the heavy chain constant region of the vaccinia virus murine monoclonal antibody is shown in SEQ ID NO.7 of the sequence listing, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.8 of the sequence listing.
[0019] The heavy chain amino acid sequence of the vaccinia virus murine monoclonal antibody is shown in SEQ ID NO.5 of the sequence listing, and the light chain amino acid sequence is shown in SEQ ID NO.6 of the sequence listing.
[0020] The mouse monoclonal antibody against poxvirus specifically binds to the H3L protein of poxvirus, thereby inhibiting viral infection of cells and providing protection.
[0021] The method for screening murine monoclonal antibodies against poxvirus using monkeypoxvirus H3L protein as an antigen mainly includes the following steps:
[0022] (1) Using H3L protein as a protein vaccine, Balb / c mice were immunized to generate a specific immune response.
[0023] (2) The spleen cells of Balb / c mice immunized with H3L were fused with myeloma cells SP2 / 0 and the clones were tested to obtain a hybridoma cell line with high affinity for H3L.
[0024] (3) Obtain mRNA from hybridoma cells, reverse the process to obtain cDNA, amplify the antibody sequence using PCR, and sequence the antibody to determine its type and sequence.
[0025] (4) Culture H3L hybridoma cells and obtain anti-H3L monoclonal antibody H489 from the cell supernatant;
[0026] Example 1: Preparation of monkeypox virus H3L protein
[0027] Given the role of H3L in poxvirus infection, we compared the extracellular amino acid sequences of the H3L protein in smallpox virus, mousepox virus, vaccinia virus, and monkeypox virus. We found that the H3L protein is highly conserved among poxviruses, with a homology of 96.9%. Figure 1 As shown.
[0028] Subsequently, pET28a(+) was selected as the expression vector. The extracellular region sequence of monkeypox virus H3L protein was synthesized and cloned into the pET28a(+) vector to obtain the expression plasmid. This plasmid was transformed into BL21 E. coli competent cells, and expression was induced by IPTG. Inclusion bodies were collected, and then the inclusion bodies were refolded by dilution. Finally, the refolded inclusion bodies were purified by Ni column chromatography, and the protein purity was identified by SDS-PAGE. Figure 2 As shown, purified H3L protein was obtained.
[0029] Example 2: Immunization of mice with H3L protein and preparation of hybridoma cell lines
[0030] H3L protein was emulsified with Freund's complete adjuvant to create a protein vaccine, which was then used to immunize healthy female Balb / c mice aged 6-8 weeks. Immunization was administered at weeks 0, 3, and 10, for a total of three immunizations. The specific method was as follows: 100 μg of H3L protein was thoroughly mixed with 100 μL of Freund's adjuvant and emulsified before being injected subcutaneously into Balb / c mice at multiple sites, 50 μL at each site, for a total injection of 200 μL. Three days before hybridoma fusion, a pulse immunization of 100 μg of H3L protein per mouse was administered.
[0031] Spleens were harvested from immunized Balb / c mice. The spleen was ground, cells were collected, centrifuged, and washed twice with culture medium. Red blood cells were lysed using erythrocyte lysis buffer. After lysis, the cells were washed twice, counted, and myeloma cells SP2 / 0 were mixed with spleen cells at a 1:3 ratio. The mixture was then fused with PEG, resuspended in HAT medium, and seeded into 40 96-well cell culture plates. The medium was changed after 6-7 days. ELISA was performed one day after medium change. OD values were... 450 >0.5 was defined as a positive clone. After three rounds of antigen-specific screening, hybridoma cell lines specific to the H3L antigen were obtained.
[0032] A hybridoma cell line with high affinity for H3L protein was obtained and cultured in RPMI-1640 medium containing 10% fetal bovine serum for 72-96 h in a cell culture incubator at 37°C and 5% CO2. Cell supernatant was collected and the monoclonal antibody secreted in the supernatant was detected. This antibody was named H489.
[0033] Example 3: Western blot identification of monoclonal antibody H489
[0034] The cell supernatant from Example 2 was separated by SDS-PAGE electrophoresis and then transferred onto a PVDF membrane using a wet transfer method. The membrane was blocked with 5% skim milk powder at room temperature for 2 hours. It was washed 5 times with TBST buffer, 5 minutes each time. The membrane was incubated with a 1:5000 dilution of goat anti-mouse HRP at room temperature for 1 hour, followed by 5 washes with TBST buffer. Finally, the membrane was observed using ECL substrate chromogenic solution. Figure 3 As shown, the supernatant secreted by this hybridoma cell line contains H489 monoclonal antibody, with the heavy chain at approximately 55 kDa and the light chain at approximately 25 kDa.
[0035] Example 4: Amplification of H489 antibody light and heavy chain sequences
[0036] The hybridoma cell line from Example 2 was cultured, and RNA was extracted from the hybridoma cell line using the following method: The sample was collected into a 1.5 mL centrifuge tube, centrifuged at 3500 rpm, the supernatant was discarded, and the cells were washed with room temperature phosphate-buffered saline (PBS) and centrifuged again; 1 mL of Trizol was added to the sample and incubated on ice for 5 min; 200 μL of chloroform was added, vortexed for 30 s, incubated on ice for 5 min, and then centrifuged at 12000 rpm; after centrifugation, the upper aqueous phase was aspirated into a new centrifuge tube, an equal volume of pre-chilled isopropanol was added, and the sample was incubated at -20℃ for 10 min; centrifuged at 12000 rpm and 4℃ for 20 min, the supernatant was discarded, and the sample was washed with pre-chilled 75% ethanol and centrifuged at 7500 rpm and 4℃ for 5 min; the ethanol was discarded, the sample was dried at room temperature for 5 min, and the RNA was dissolved in DEPC water. The obtained RNA was then used as a template for reverse transcription using a reverse transcription kit, with the following reaction conditions: 65℃ for 5 min, 4℃ for 5 min. This product was then used as a template for a second round of amplification, with the following reaction conditions: 30℃ for 10 min; 42℃ for 60 min; 70℃ for 15 min; 4℃ for 5 min. PCR amplification was then performed using universal primers for mouse antibodies, with the following reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 58℃ for 1 min; 72℃ for 1 min; 72℃ for 5 min; 4℃ for 5 min, with the second to fourth steps repeated 30 times. The DNA was then detected using DNA gel electrophoresis. Figure 4 As shown, the heavy and light chain sequences of the H489 monoclonal antibody were successfully amplified. The heavy chain sequence is approximately 1500 bp, and the light chain sequence is approximately 750 bp. The amplified sequences were sent for sequencing, and the antibody sequence was confirmed through analysis and alignment. Sequence verification was performed using vbase2 (http: / / www.vbase2.org / ). The heavy chain type of this antibody is IgG2b, and the V region belongs to the IgH-V7183VH5 family; the light chain type is Igκ, and the V region belongs to the IgKV21 family. The antibody type is IgG2b, and this antibody is named H489.
[0037] The amino acids in the heavy chain variable region have the sequence described in SEQ ID NO.1 of the sequence listing, and the nucleotides have the sequence described in SEQ ID NO.2 of the sequence listing. The amino acids in the light chain variable region have the sequence described in SEQ ID NO.3 of the sequence listing, and the nucleotides have the sequence described in SEQ ID NO.4 of the sequence listing.
[0038] The constant regions of the heavy and light chains have the sequences described in SEQ ID NO.7 and SEQ ID NO.8 of the sequence listing.
[0039] The amino acids of the heavy and light chains have the sequences described in SEQ ID NO.5 and SEQ ID NO.6 of the sequence listing.
[0040] Example 5: Detection of H489 antibody binding specificity
[0041] 1. The specific binding of monoclonal antibodies to monkeypox virus H3L protein was detected by ELISA. The specific procedure is as follows: Monkeypox virus H3L protein, M1R protein, or BSA (bovine serum albumin) was coated onto the ELISA plate at a concentration of 5 μL / well for each protein, and the plate was incubated overnight at 4°C. The next day, the plate was blocked with 1% BSA at 37°C for 2 h. Hybridoma cell culture supernatant collected in Example 2 at different dilutions was added to each well, and the plate was incubated at 37°C for 2 h. The plate was washed four times with TBST buffer. Then, the plate was incubated with HRP-labeled anti-mouse antibody secondary antibody at 37°C for 1 h. The plate was washed four times with TBST buffer. 60 μL / well of TMB was added for color development, and the reaction was stopped after 15 min by adding 60 μL / well of dilute hydrochloric acid. The plate was then analyzed using a microplate reader.
[0042] ELISA results: The H489 monoclonal antibody showed a high binding affinity to the monkeypox virus H3L protein, with the binding activity remaining higher than the control group even when the H489 supernatant was diluted 21870-fold. Simultaneously, the H489 monoclonal antibody did not bind to the monkeypox virus M1R protein or BSA. These results indicate that H489 specifically binds to the H3L protein, without binding to other irrelevant proteins. Therefore, H489 is a specific monoclonal antibody against monkeypox virus H3L, exhibiting excellent specificity and affinity for the H3L protein.
[0043] Table 1. Comparison of binding activity between H489 monoclonal antibody and monkeypox virus H3L protein.
[0044]
[0045] 2. H489 monoclonal antibody binding activity against monkeypox virus M1R protein control table
[0046]
[0047] Table 3. Comparison of binding activity between H489 monoclonal antibody and monkeypox virus BSA protein
[0048]
[0049] 2. The binding of monoclonal H489 antibody to poxvirus was detected by ELISA. The specific procedure is as follows: The ELISA plate was coated with vaccinia virus (VACV), mousepox virus (ECTV), or H1N1 influenza virus (PR8). The coating dose for each virus was 1×10⁻⁶. 7 PFU / mL, 100 μL / well for coating, reacted overnight at 4°C, and the subsequent steps were the same as in Example 5.
[0050] ELISA results showed that the H489 monoclonal antibody exhibited good binding affinity to vaccinia virus (a member of the orthopoxvirus genus) and mousepox virus, but not to influenza virus. This indicates that the H489 monoclonal antibody specifically binds to poxviruses, but not to other unrelated viruses. Therefore, the H489 monoclonal antibody can be used as a poxvirus detection tool, specifically targeting orthopoxviruses such as vaccinia virus and mousepox virus.
[0051] Table 4. Binding activity comparison table of H489 monoclonal antibody and vaccinia virus (VACV)
[0052]
[0053] Table 5. Comparison of binding activity between H489 monoclonal antibody and mousepox virus (ECTV)
[0054]
[0055] Table 6. Binding activity comparison table of H489 monoclonal antibody and H1N1 influenza virus (PR8)
[0056]
[0057] Example 6: Detection of the neutralizing effect of H489 antibody
[0058] The specific operation method is as follows: BSC-1 cells in 6-well plates were infected with vaccinia virus. H489 monoclonal antibody supernatant diluted 5-fold or 50-fold was added to the experimental group. RPMI-1640 medium containing 2.5% fetal bovine serum was used. After culturing in a 37°C, 5% CO2 incubator for 2-3 days, the cells were fixed with 4% paraformaldehyde. After crystal violet staining, the cell morphology was observed and the number of plaques was counted.
[0059] Neutralization experiment results as follows Figure 5As shown, NC represents wells without virus infection, with dense cells and no plaques; con represents the control wells with virus infection, where virus infection caused numerous plaques and comet tails; H489 1 / 5 and H489 1 / 50 represent wells infected with the virus and supplemented with different dilutions of H489 supernatant. The addition of 5-fold or 50-fold diluted H489 monoclonal antibody significantly inhibited viral infection and replication, significantly reduced the number of plaques, and suppressed comet tail formation. The neutralization efficiency of the 5-fold diluted H489 was over 90%.
[0060] Table 7. Comparison of Neutralization Efficacy Detection of H489 Antibody
[0061]
[0062] Therefore, H489 monoclonal antibody has good neutralizing activity and can effectively inhibit viral infection and replication in cells, and has application value in detecting poxvirus infection and preventing poxvirus infection.
Claims
1. A murine monoclonal antibody to a poxvirus characterized in that, The amino acid sequence of the heavy chain variable region domain is shown in SEQ ID NO. 1 of the Sequence Listing, and the amino acid sequence of the light chain variable region domain is shown in SEQ ID NO. 3 of the Sequence Listing.
2. The murine monoclonal antibody to poxvirus according to claim 1, characterized in that, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 7 of the Sequence Listing, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 8 of the Sequence Listing.
3. The murine monoclonal antibody to poxvirus according to claim 1, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO. 5 of the Sequence Listing, and the amino acid sequence of the light chain is shown in SEQ ID NO. 6 of the Sequence Listing.
4. The murine monoclonal antibody to poxvirus according to claim 1, characterized in that, The heavy chain variable region domain belongs to IgH-V7183 VH5 family.
5. The murine monoclonal antibody to poxvirus according to claim 1, characterized in that, The light chain variable region domain belongs to IgKV21 family.
6. The vaccinia virus murine monoclonal antibody according to claim 3, characterized in that, The heavy chain type is IgG2b; and the light chain type is IgK.
7. Use of the poxvirus murine monoclonal antibody according to any one of claims 1-6 in the preparation of a medicament for preventing or treating anti-poxvirus infection. The poxvirus includes: The poxvirus is vaccinia virus or monkeypox virus.
8. Use of the poxvirus murine monoclonal antibody according to any one of claims 1-6 in the preparation of a detection agent for poxvirus. The poxvirus is vaccinia virus or monkeypox virus.