CN113861285B

CN113861285B - Humanized monoclonal antibody of poxvirus and application thereof

Info

Publication number: CN113861285B
Application number: CN202111078840.6A
Authority: CN
Inventors: 方敏; 顾秀玲; 张毓凡; 姜威; 卢娇; 顾光磊
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2021-09-15
Filing date: 2021-09-15
Publication date: 2023-03-10
Anticipated expiration: 2041-09-15
Also published as: CN113861285A

Abstract

The invention discloses a pox virus humanized monoclonal antibody and application thereof, belonging to the technical field of medicines. The invention takes vaccinia virus surface membrane antigen A33 protein expressed by escherichia coli as antigen, a memory B cell with A33 protein specificity is screened from PBMCs of a volunteer inoculated with smallpox vaccine, then the specific B cell is inverted and amplified to obtain a variable region segment of an antibody, the variable region segment is further connected with a constant region into an expression vector, and a series of functional tests are carried out after mammalian cell expression and purification to obtain a human monoclonal antibody with the function of protecting smallpox virus. The antibody has better binding capacity with antigen detected by ELISA, and has higher protective effect in vitro neutralization test and mouse infection. The humanized antibody has the application value of clinically treating and preventing smallpox virus.

Description

Humanized monoclonal antibody of poxvirus and application thereof

Technical Field

The invention belongs to the technical field of medicines.

Background

Smallpox (Smallpox) is a virulent infectious disease caused by Smallpox virus (Variola virus) and is by far the only infectious disease that has been eradicated worldwide by humans. Variola virus belongs to the genus Orthopoxvirus (Orthopoxvirus) which also includes Vaccinia virus (vacciia virus, VACV, used as a vaccine for smallpox), monkeypox virus (monkeypoxy) and mousepox virus (ECTV), among others, which are highly homologous. Monkeypox virus or other poxviruses that have been genetically engineered may infect humans across species. Since mass immunization has been stopped for nearly 40 years, most of the population is not immune to smallpox virus at present. Therefore, it is necessary to construct humanized antibodies against poxviruses with high affinity and broad specificity, which can provide rapid detection of poxviruses and provide strategic antibody reserves for immunotherapy of diseases.

Orthopoxvirus infects host cells in two forms of infection: intracellular Mature Virions (IMV) and Extracellular Evolved Virions (EEV). The A33 protein is an important protein on the envelope of its extracellular virion EEV, which is a protein encoded by the A33R gene in the genome of orthopoxviruses and is expressed later in infection. The A33 protein is an important protein in the migration process of virus infected host cells, and researches show that the outer membrane protein A33 of vaccinia virus in orthopoxvirus is an important target of natural antibody response of anti-poxvirus. By the mechanism of antigen-antibody reaction, memory B cells specific for an antigen can be screened by the antigen. Compared with murine monoclonal antibodies or chimeric antibodies, the monoclonal antibodies lack the defects of cell-mediated cytotoxicity, complement-dependent cytotoxicity and the like, so that the curative effect is reduced and even anaphylactic reaction is caused, and the sorting of specific memory B cells by using A33 as an antigen is a very effective method for developing humanized neutralizing antibodies aiming at the poxvirus.

Disclosure of Invention

In order to solve the problems, the invention uses A33 protein as antigen, then selects B lymphocyte capable of specifically binding A33 from Peripheral Blood (PBMCs) of a volunteer inoculated with smallpox vaccine by flow sorting, then carries out RT-PCR and PCR amplification on the single B lymphocyte, thereby obtaining variable region of light and heavy chains of the antibody, further connects the variable region with constant region into an expression vector, and detects the function of the antibody after expression and purification in mammalian cells, including the binding condition with A33, in vitro neutralization experiment.

The amino acid sequence of a heavy chain variable region of the poxvirus humanized monoclonal antibody provided by the invention is SEQ ID NO.1, and the amino acid sequence of a light chain variable region is SEQ ID NO.3.

In a specific embodiment of the present invention, the constant region sequences of the heavy and light chains of the poxvirus humanized monoclonal antibody are as shown in SEQ ID No.7 and SEQ ID No. 8.

In a specific embodiment of the present invention, the amino acid sequences of the heavy and light chains of the poxvirus humanized monoclonal antibody are as shown in SEQ ID No.5 and SEQ ID No. 6.

In a specific embodiment of the present invention, the poxvirus human monoclonal antibody specifically binds to poxvirus a33 protein to inhibit the infection of cells by poxviruses, thereby protecting the cells.

The invention also provides a method for screening the humanized monoclonal antibody of the A33 antigen against the smallpox virus, which comprises the following steps:

first, a recombinant plasmid containing a code a33 was constructed using pET28a (+) as an expression vector, and the recombinant plasmid was transformed into DH5 α competent cells for expression and purification.

Screening specific B cells from peripheral blood of a volunteer inoculated with the smallpox virus vaccine by taking A33 as an antigen to obtain an antibody light and heavy chain variable region sequence, firstly constructing a single-chain antibody, and further screening by ELISA.

(3) And (3) constructing the determined single-chain antibody into a complete antibody IgG form, transfecting the plasmid into 293 cells, and purifying to obtain the humanized antibody.

In a particular embodiment of the invention, the method further comprises performing an in vitro neutralization assay on the screened antibody.

The antibody obtained by screening is a neutralizing antibody capable of effectively inhibiting the invasion of the poxvirus, and has higher affinity to A33, so the antibody not only can be applied to clinical monitoring, but also can be used for protecting the poxvirus.

The invention has the beneficial effects that:

the invention carries out flow sorting by constructing the A33R body antigen, greatly increases the sorting accuracy by multi-fluorescence labeling, reduces the heterogeneity of the antibody by the humanized monoclonal antibody, can be applied to the preparation of other virus antibodies, and has universal applicability.

Drawings

FIG. 1: a33 protein expression SDS-PAGE picture.

FIG. 2 is a schematic diagram: flow sort variola virus-specific B lymphocyte maps.

FIG. 3: antibody variable region sequence amplification map.

FIG. 4 is a schematic view of: single chain antibody expression profiles.

FIG. 5: ELISA, detection of the single-chain antibody and A33 binding map.

FIG. 6: the surface plasmon resonance technology detects the binding pattern of the single-chain antibody and A33.

FIG. 7: single chain antibody in vitro neutralization assay contrast.

FIG. 8: data comparison of in vitro neutralization assays for single chain antibodies.

FIG. 9: h2IgG expression purification and detection of binding to a 33.

FIG. 10: h2 Graph of IgG protection of mice against infection by vaccinia virus.

Detailed Description

The invention takes A33 protein as antigen, then selects B lymphocyte which can specifically bind A33 from Peripheral Blood (PBMCs) of a volunteer inoculated with smallpox vaccine by flow sorting, then carries out RT-PCR and PCR amplification on single B lymphocyte to obtain variable region of light and heavy chain of antibody, further connects the variable region and the constant region to an expression vector, and detects the function of the antibody after expression and purification in mammalian cells, including the binding condition with A33 and in vitro neutralization experiment.

The amino acid sequence of the heavy chain variable region of the poxvirus humanized monoclonal antibody is SEQ ID No.1, the amino acid sequence of the light chain variable region is SEQ ID No.3, and the antibody is named H2.

The constant region sequences of the heavy chain and the light chain of the poxvirus humanized monoclonal antibody are shown as SEQ ID NO.7 and SEQ ID NO. 8.

The amino acid sequences of the heavy chain and the light chain of the poxvirus humanized monoclonal antibody are shown as SEQ ID NO.5 and SEQ ID NO. 6.

The poxvirus humanized monoclonal antibody specifically binds to poxvirus A33 protein, thereby inhibiting the infection of cells by the virus and protecting the cells.

A33 is used as an antigen to screen humanized monoclonal antibodies of the anti-smallpox virus, and the method mainly comprises the following steps:

constructing a recombinant plasmid containing a code A33 by taking pET28a (+) as an expression vector, and transforming the recombinant plasmid into DH5 alpha competent cells for expression and purification;

screening specific B cells from peripheral blood of a volunteer inoculated with the smallpox virus vaccine by taking A33 as an antigen to obtain an antibody light and heavy chain variable region sequence, firstly constructing a single-chain antibody, and further screening by ELISA;

Example 1:

expression purification of vaccinia virus outer Membrane protein A33 protein in Orthopoxvirus

In view of the role of a33 in poxvirus infection, we aligned the extracellular amino acid sequence of a33 in variola virus, murine poxvirus, vaccinia virus, monkeypox virus, a33 is highly conserved in poxviruses with homology as high as 92.48%, which also demonstrates that a33 is an important target for natural antibody responses against poxviruses.

Then selecting pET28a (+) as an expression vector, selecting a position between two enzyme cutting sites of NcoI and BamHI as an insertion site of an A33 protein gene to obtain a recombinant plasmid, transforming the recombinant plasmid into BL21 escherichia coli competent cells, adding IPTG (isopropyl-beta-thiogalactoside) for induction when OD600 of bacterial liquid is between 0.6 and 0.8, collecting inclusion bodies after about 3 to 4 hours, renaturing the inclusion bodies through a dilution method, and finally purifying the renatured inclusion bodies through a Ni column and identifying the protein purity through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) as shown in figure 1.

Sorting of poxvirus-specific memory B lymphocytes

Lymphocytes were isolated from PBMCs from smallpox vaccinated volunteers and resuspended to 10 ⁷ At a density of/ml and mixed with A33 and anti-human PE-CD19 (Biolegend, 302208), anti-human FITC-IgM (Biolegend, 314506), anti-human percp/Cy5.5-CD27 (Biolegend, 124214) and incubated at 4 ℃ for 30min, washed once with PBS, and the cells were resuspended in 300uL 1640 medium in sterile sorting tubes. IgM-, CD19+ and CD27+ single cells were collected by FACSAria III sorting in a 96-well plate to obtain B lymphocytes, as shown in FIG. 2.

Antibody sequence variable region amplification and single-chain antibody construction

The resulting B lymphocytes were reverse transcribed by reverse transcriptase III, the reverse transcription primers are shown in Table 1.

TABLE 1 reverse transcription primers

Carrying out PCR by taking the reverse transcription product as a template, and designing a corresponding primer, wherein the reaction is as follows: 94 ℃ for 2min; 35 cycles of 30s at 98 deg.C, 1min at 55 deg.C, and 30s at 68 deg.C for 1min; then 68 ℃ for 5min. Then the product of the second round is used as a template for carrying out the second round of amplification, and the reaction conditions are as follows: 94 ℃ for 2min; 30s at 98 deg.C and 1min at 60 deg.C; 35 cycles of 1min at 68 ℃ for 30 s; then 68 ℃ for 5min. The amplification products were detected by DNA gel electrophoresis as shown in FIG. 3.

The amplified sequence is sent to sequence, and the variable sequence of the antibody is analyzed through software to construct a single-chain antibody, which is designed as follows: SP-VL-Linker-VH-3X Flag.

Wherein the amino acid sequence of the Leader sequences is described in SEQ ID NO.9, and the amino acid sequence of the Linker is described in SEQ ID NO. 10.

The amino acid of the heavy chain variable region has a sequence shown by SEQ ID NO.1 in a sequence table and the nucleotide has a sequence shown by SEQ ID NO.2 in the sequence table, and the amino acid of the light chain variable region has a sequence shown by SEQ ID NO.3 in the sequence table and the nucleotide has a sequence shown by SEQ ID NO.4 in the sequence table.

The constant regions of the heavy chain and the light chain have the sequences shown in SEQ ID NO.7 and SEQ ID NO.8 in the sequence table.

The amino acids of the heavy chain and the light chain have the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence table.

This antibody is designated H2.

Example 2:

performance testing of Single chain antibodies

First ELISA for detecting the binding of a single-chain antibody to a33

To verify whether the screened antibodies specifically bind to a33 and are functional, they were tested by ELISA and in vitro neutralization assays.

The single-chain antibody plasmid is transfected into 293T cells, and after 24h, the supernatant is collected, and the expression of the single-chain antibody plasmid is detected by Western method, as shown in figure 4. The A33 protein was diluted with the coating solution and coated on a 96-well plate, and then binding was detected by a single-chain antibody supernatant, from which H2 single-chain antibodies were selected, as shown in FIG. 5.

The combination of the detection of the surface plasmon resonance technology and the A33

Surface plasmon resonance analysis was performed using Biacore T100 (Biacore Inc).

The method comprises the following specific steps: firstly, an H2 single-chain antibody (ScFv) is fixed on a CM5 chip in an amino coupling mode, the fixed quantity is controlled at 4800RU, H2 single-chain antibody expression supernatant is combined in an antibody capture mode, the flow rate is 30ul/min, sample injection is carried out for 60s, dissociation is carried out for 180s, glycine with the regeneration condition of PH2.0 is set for one time repetition, the concentration is from 62.5uM to 1000uM,250uM is set, and the calculation of a binding kinetic constant is analyzed by utilizing BIAevaluation software T100 (Biacore Inc) software.

The SPR results showed that the affinity of the H2 single chain antibody to A33 was 59.4nM, as shown in FIG. 6.

Abs	Ka(1/Ms)	Kd(1/s)	KD(M)
				H2 ScFv	3.33E+04	0.001976	5.94E-08

The experiments in the nature of the stone

The BSC-1 cells of a 6-well plate are infected by a murine pox virus (Ectromlia virus, ECTV), 10-fold dilution and 50-fold dilution of an H2 single-chain antibody and 100-fold and 500-fold dilution of anti-A33 mouse serum are respectively added into experimental groups, after 5 days of infection, the experimental groups are fixed by 4% paraformaldehyde, and the comet tail condition is observed by crystal violet staining, as shown in figure 7, figure 8 is a statistical chart, the H2 single-chain antibody can inhibit the propagation of the murine pox virus in the BSC-1 cells and inhibit the formation of the comet tail, and the neutralization efficiency reaches over 90%.

Example 3:

h2 Expression purification of IgG antibodies

The light and heavy chain variable region sequences SEQ ID NO.1 and SEQ ID NO.3 of the signal peptide sequences SEQ ID NO.9 and H2 and the constant region sequences SEQ ID NO.7 and SEQ ID NO.8 are spliced together to construct a complete H2IgG form. The constructed H2IgG plasmid is transfected into 293F cells, cell supernatant is collected after 3 days of culture, H2IgG antibody is obtained after purification through protein A, and finally the purification condition is verified through polyacrylamide gel electrophoresis, as shown in figure 9.

Example 4:

animal protection experiment

Female BALB/c mice (Wintonly Hua) at 6-8 weeks, 5 in one group. Intraperitoneal injection of 1X 10 per mouse ⁶ PFU vaccinia virus, the survival and weight change of mice were recorded within 15 days. 48 hours after infection, H2IgG antibody was injected at a dose of 22mg/kg, PBS was injected at an equal dose to the control group, and H2 antibody was injected to the control groupThe protective efficiency was as high as 80% (fig. 10A, B) and the percent change in body weight of the mice is shown in table 2.

TABLE 2 percent weight change of mice

Although the above examples are merely examples for clearly illustrating the present invention, the present invention is not limited to the embodiments. Various changes and modifications can be made without departing from the spirit and scope of the invention, and it is intended that all such obvious changes and modifications as may be resorted to herein be included within the scope of the invention.

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Claims

1. The poxvirus humanized monoclonal antibody is characterized in that the amino acid sequence of the heavy chain variable region structural domain is shown as the sequence shown in SEQ ID NO.1 in the sequence table, and the amino acid sequence of the light chain variable region structural domain is shown as the sequence shown in SEQ ID NO.3 in the sequence table.

2. The poxvirus humanized monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain has the sequence given in SEQ ID No. 5.

3. The poxvirus humanized monoclonal antibody of claim 1, wherein the amino acids of the light chain have the sequence as set forth in SEQ ID No.6 of the sequence listing.

4. The poxvirus humanized monoclonal antibody of claim 1, further comprising a heavy chain constant region comprising the amino acids set forth in SEQ ID No.7 of the sequence listing and a light chain constant region comprising the amino acids set forth in SEQ ID No.8 of the sequence listing.

H2IgG antibodies comprising the poxvirus human monoclonal antibodies of claims 1 to 4.

6. Use of an H2IgG antibody according to claim 5 in the manufacture of a medicament for the detection, prevention or treatment of an anti-poxvirus comprising: smallpox virus, murine pox virus, bean virus, monkey pox virus.